Interferon-gamma-binding molecules for treating septic shock, cachexia, immune diseases and skin disorders

ABSTRACT

The present invention concerns molecules which bind and neutralize the cytokine interferon-gamma. More specifically, the present invention relates to sheep-derived antibodies and engineered antibody constructs, such as humanized single-chain Fv fragments, chimeric antibodies, diabodies, triabodies, tetravalent antibodies, peptabodies and hexabodies which can be used to treat diseases wherein interferon-gamma activity is pathogenic. Examples of such diseases are: septic shock, cachexia, multiple sclerosis and psoriasis.

This application is a section 371 national stage filing ofPCT/EP98/05165, filed Aug. 14, 1998 (published in English on Feb. 25,1999 As WO 99/09055) and claiming priority to EP 97870122.5 filed Aug.18, 1997; and EP 98870139.7 filed Jun. 18, 1998.

FIELD OF THE INVENTION

The present invention concerns molecules which bind and neutralize thecytokine interferon-gamma. More specifically, the present inventionrelates to sheep-derived antibodies and engineered antibody constructs,such as humanized single-chain Fv fragments, chimeric antibodies,diabodies, triabodies, tetravalent antibodies and peptabodies which canbe used to treat diseases wherein interferon-gamma activity ispathogenic. Examples of such diseases are: septic shock, cachexia,multiple sclerosis and psoriasis.

BACKGROUND OF THE INVENTION

Interferon-gamma (IFNγ) is a member of the interferon family ofimmunomodulatory proteins and is produced by activated T helper type-1cells (Th1 cells) and natural killer cells (NK cells). Apart from itspotent antiviral activity, IFNγ is known to be involved in a variety ofimmune functions (for a review, see Billiau, 1996) and inflammatoryresponses. Indeed, IFNγ is the primary inducer of the expression of themajor histocompatibility complex (MHC) class-II molecules (Steinman etal., 1980) by macrophages and other cell types and stimulates theproduction of inflammatory mediators such as tumor necrosis factor-alpha(TNFα), interleukin-1 (IL-1) and nitric oxide (NO) (Lorsbach et al.,1993). In this respect, IFNγ is shown to be important in themacrophage-mediated defence to various bacterial pathogens. Furthermore,IFNγ is also shown to be a potent inducer of the expression of adhesionmolecules, such as the intercellular adhesion molecule-1 (ICAM-1, Dustinet al., 1988), and of important costimulators such as the B7 moleculeson professional antigen presenting cells (Freedman et al., 1991).Moreover, IFNγ induces macrophages to become tumoricidal (Pace et al.,1983) and provokes Ig isotype switching (Snapper and Paul, 1987).

The anti-viral, tumoricidal, inflammatory- and immunomodulatory activityof IFNγ clearly has beneficial effects in a number of clinicalconditions. However, there are a number of clinical situations in whichIFNγ-activity has deleterious effects. These include cancer cachexia(Denz et al., 1993; Iwagaki et al., 1995), septic shock (Doherty et al.,1992), skin disorders such as psoriasis and bullous dermatoses (Van denOord et al., 1995), allograft rejection (Landolfo et al., 1985;Gorczynski, 1995), chronic inflammations such as ulcerative colitis andCrohn's disease (WO 94/14467 to Ashkenazi & Ward), and autoimmunediseases such as multiple sclerosis (MS, Panitch et al., 1986),experimental lupus (Ozmen et al., 1995), arthritis (Jacob et al., 1989;Boissier et al., 1995) and autoimmune encephalomyelitis (Waisman et al.,1996).

Cachexia is a phenomenon often seen in cancer patients and is associatedwith losses of lean body mass, and altered carbohydrate and lipidmetabolism. This so called ‘chronic wasting syndrome’ is often theimmediate cause of death. In recent years, interest has focused on therole of proinflammatory cytokines in cancer related cachexia. Currentdata support the concept that cachexia is linked to the presence ofcertain cytokines among which IFNγ seems to play a central role. Denz etal. (1993) reported that increased neopterin and decreased tryptophanconcentrations—which are closely related to IFNγ-activity—are detectedin cachectic patients suffering from hematological disorders. Neopterinis synthesized and secreted by monocytes/macrophages upon stimulation byIFNγ from activated T cells. Tryptophan is an indispensable amino acidwhich can be catabolized by indoleamine 2,3-dioxygenase, an enzymeinduced by IFN's, and which absence initiates mechanisms responsible forcachexia (Brown et al., 1991). The correlation between high neopterinlevels, decreased tryptophan levels and weight loss was confirmed byIwagaki et al. (1995). In experimental models, cancer-induced cachexiacan be altered by the administration of IFNγ neutralizing antibodies(Matthys et al., 1991; Langstein et al., 1991)

Septic shock is the result of a severe bacterial infection, and remainsa common cause of death among critically ill, hospitalized patientsdespite improvements in supportive care (Bone et al., 1992). Althoughseptic shock may be associated with gram-positive infections, attentionhas focused on the more common pathogenesis of gram-negative sepsis andthe toxic role of endotoxin (=lipopolysaccharide or LPS), a component ofthe outer membrane of gram-negative and some gram-positive bacteria.Many of the effects of LPS are mediated through the release of cytokinessuch as TNFα (Tracey, 1991), IL-1 (Wakabayashi et al., 1991) and IFNγ(Bucklin et al., 1994). Much of the evidence supporting the role ofthese cytokines as mediators of septic shock comes from lethalitystudies involving the blockade of individual cytokines, resulting inprotection of experimental animals from otherwise lethal doses ofendotoxin or gram-negative bacteria. One of the first events in septicshock is the activation of T cells by antigen presenting cells ontowhich bacterial superantigen is bound (Miethke et al., 1993). Uponactivation, for which co-stimulation of CD28 is essential (Saha et al.,1996), these T cells proliferate and produce a surge of proinflammatorycytokines such as IL-2, TNFα and IFNγ eventuating in the clinicalsyndrome. Also, it is hypothesized that LPS induces the expression ofthe α1/β1 integrin (VLA-1) heterodimer on activated monocytes which thendisplay an increased capacity to adhere to the endothelial basementmembrane. Similar effects can be induced by incubation of monocytes withIFNγ (Rubio et al., 1995). VLA-1 might also contribute to furthermonocyte activation and potentiation of the production ofmonocyte-derived pro-inflammatory cytokines during sepsis (Rubio et al.,1995). Although very promising results were obtained with antibodiesneutralising TNFα in experimental animal models, clinical trials withanti-TNFα antibodies revealed only a slight reduction or even noreduction in mortality rate of patients with septic shock (Wherry etal., 1993; Reinhart et al., 1996). A fusion protein containing theextracellular portion of the TNF receptor and the Fc portion of IgG1also did not affect mortality (Fisher et al., 1996). Pentoxifylline(PTX), a methyl xanthine derivative, is currently being tested for itseffect on the outcome of septic shock. PTX is known to lower the serumconcentrations of at least TNFβ, IL-1 and IFNα (Bienvenu et al., 1995;Zeni et al., 1996). Initial data reveal that PTX leads to an improvementof the clinical status of septic patients (Mándi et al., 1995). There isevidence that IFNγ is a mediator of lethality during sepsis. Antibodiesthat either neutralize IFNγ or block the IFNγ-receptor are protectingagainst lethality (Bucklin et al., 1994; Doherty et al., 1992). Asynergistic effect between IFNγ and TNFα has also been suggested(Doherty et al., 1992; Ozmen et al., 1994). Although not in itselflethal, IFNγ has been shown to be essential for the manifestation ofTNF-induced lethality in the generalized Shwartzman reaction (Ozmen etal., 1994).

Bullous, inflammatory and neoplastic dermatoses are a heterogenous groupof skin disorders during which IFNγ may play a pathogenic role. Bullousdermatoses encompass epidermolysis bullosa acquisita, bullous pemhigoid,dermatitis herpetiformes Duhring, linear IgA disease, herpesgestationis, cicatricial pemhigoid, bullous systemic lupuserythematosis, epidermolysis bullosa junctionalis, epidermolysis bullosadystrophicans, porphyria cutanea tarda and Lyell-Syndrome (Megahed,1996). Also erythema exsudativum multiform major (Kreutzer et al.,1996), IgG-mediated subepidermal bullous dermatosis (Chan & Cooper,1994), bullous lichen planus (Willsteed et al., 1991) and paraneoplasticbullous dermatosis (Pantaleeva, 1990) can be classified among thebullous dermatoses. A pathogenic role of IFNγ during bullous dermatoseshas been suggested by Van den Oord et al. (1995). The role of IFNγduring inflammatory and neoplastic dermatoses, compared to bullousdermatoses, has been more extensively investigated. Indeed, it has beendemonstrated that IFNγ is involved during the pathogenesis of verrucosis(Asadullah et al., 1997), eosinophilic pustular folliculitis (Teraki etal., 1996), cutaneous T cell lymphoma (Wood et al., 1994), granulomafaciale (Smoller & Bortz, 1993), Sweet's syndrome (Reuss-Borst et al.,1993), atopic eczema (Arenberger et al., 1991) , follicular mucinosis(Meisnerr et al., 1991), lichen-planus and psoriasis (Vowels et al.,1994). One of the most extensively studied inflammatory dermatoses ispsoriasis. Psoriasis is a hyperproliferative skin disorder affectingapproximately 2% of the population. Evidence is accumulating that thedisease has a T-cell mediated autoimmune etiology. The role of T-cellsin psoriasis has been demonstrated by Gottlieb et al. (1995). The latterauthors suggested that, in most of the patients, clinical andhistopathological features of psoriasis are primarily linked to skininfiltration by IL-2 receptor-positive leukocytes. Disease improvementcan be induced by the administration of a fusion protein composed ofhuman interleukin-2 and fragments of diphteria toxin, which selectivelyblocks the growth of activated lymphocytes. Other effectiveanti-psoriatic, T-cell suppressing agents include the immunosuppressivedrugs cyclosporin and FK506 (Griffiths, 1986) and anti-CD4 monoclonalantibodies (Morel et al., 1992). More direct evidence for the role of Tcells in the induction of the complex tissue alterations seen inpsoriasis has been generated by Schön et al. (1997) using a model withscid/scid mice in which they transferred naive, minor histocompatibilitymismatched CD4⁺ T-cells, resulting in the development of a skin disorderthat resembles psoriasis. The autoimmune character of the disease hasbeen proposed by Valdimarsson et al. (1995) who stated that products ofactivated T-cells can induce keratinocytes of individuals with psoriaticpredisposition to express determinants that are recognized by T cellsspecific for epitopes on β-haemolytic streptococci. Several data suggestthat IFNγ may play a crucial role in the pathogenesis of psoriasis.IFNγ, produced by activated T cells would be involved in the recruitmentof lymphocytes (Nickoloff, 1988), in the induction of activation andadhesion molecules on epidermal keratinocytes (Dustin et al., 1988), aswell as in the abnormal keratinocyte proliferation (Barker et al.,1993). Not only enhanced levels of IFNγ has been detected in psoriaticepidermis (Kaneko et al., 1990), also de novo suprabasal expression ofIFNγ receptor in psoriasis has been demonstrated (Van den Oord et al.,1995).

Inflammatory bowel disease (IBD), which encompasses ulcerative colitisand Crohn's disease, is characterized by the appearance of lesions ofunknown aetiology in most parts of the gut. IBD is rather common, with aprevalence in the range of 70-170 in a population of 100, 000. Thecurrent therapy of IBD involves the administration of anti-inflammatoryor immunosuppressive agents, which usually bring only partial results,and surgery. In view of the apparent shortcomings of the presenttreatment, Ashkenazi and Ward (WO 94/14467) suggested the usage of abispecific antibody construct targeting IFNγ and another molecule, suchas IL-1 and TNFα, to treat IBD. However, the exact role of IFNγ duringIBD is not well understood.

MS is a severely disabling progressive neurological disease of unknownaetiology, but probably involving autoimmune responses and resulting inthe appearance of focal areas of demyelinisation (Williams et al.,1994). MS affects 1 in 1000 persons in the USA and Europe, but due toimproved diagnosis that number is increasing. Onset of disease isusually around 30 years of age and, on average, patients are in need oftreatment for another 28 years. MS is among the most expensive chronicdiseases of western society, based on duration and intensity of care.However, diagnosis of exacerbations and early identification of onset ofexacerbations has improved greatly, allowing design of novel treatmentstrategies. Active multiple sclerosis lesions feature T-lymphocyte andmonocyte-macrophage accumulations at plaque margins where myelin isbeing destroyed. The inflammatory cells that invade the white matter andthe soluble mediators that they release are held primarily responsiblefor myelin breakdown. Population-based studies indicate that certainHLA-antigens occur with higher frequency in patients with MS (withpredominant MHC being the Dw2(DR2)DQ1.2 haplotype (Olerup et al., 1991).Similar associations of class I and class II haplotypes have also beendetected in other autoimmune disorders such as rheumatoid arthritis andinsulin dependent diabetes (Nepom, 1993). The lesions of MS arecomparable to those found in chronic relapsing experimental allergicencephalitis (EAE), an autoimmune disease that can be induced in animalsby immunization with e.g. whole myelin (Allen et al., 1993) or with themyelin/oligodendrocyte glycoprotein (Genain et al., 1995b). The lesionsassociated with EAE are similar in appearance as the ones occurring inMS and also contain inflammatory infiltrates of T-cells and macrophages(Genain et al., 1995b). Furthermore, in adoptive transfer experiments, Tcells sensitized to specific myelin antigens can transfer the diseasestate of EAE (Genain et al., 1995b; Waldburger et al., 1996). A fewyears ago, the American FDA approved the use of the immunosuppressivedrug interferon (trade name Betaseron) for treatment of chronicrelapsing MS. The effect of this drug—although modest—clearlydemonstrates the involvement of the cytokine network in thepathophysiology of MS. In the last few years, a large number of studieshave addressed the molecular mechanism by which Betaseron exerts itsbeneficial effects. Lately, it was shown that IFNβ dose-dependentlyinhibited T-cell proliferation, expression of IL-2 receptors andsecretion of IFNγ, TNFα and IL-13 (Rep et al., 1996). Furthermore, itwas demonstrated that IFNβ could specifically prevent the IFNγ-inducedup regulation of MHC class II antigens and adhesion molecules onantigen-presenting cells (Jiang et al., 1995) and human brainmicrovessel endothelial cells (Huynh et al., 1995).

One of the earliest events in MS is damage of the blood brain barrier(BBB) by activated, encephalitogenic T-cells (Tsukada et al., 1993). Themechanism by which these cells destruct locally the BBB, which is mainlyconstituted of endothelial cells, is not elucidated, but it is knownthat at the systemic level, local production of certain cytokines suchas IFNγ enhance the capability of lymphocytes to adhere to endothelialcells (Yu et al., 1985; Tsukada et al., 1993). Also, on choroid plexusepithelial cells of EAE animals, an increased expression of ICAM-1 andVCAM-1 (Steffen et al., 1994), for which LFA-1 and VLA-4 are the naturalligands on lymphocytes, has been observed. Mc Carron et al. (1993)reported that adhesion of MBP-specific T lymphocytes was significantlyup regulated when cerebral endothelial cells were treated with IL-1,TNFα or IFNγ. That the adhesion of encephalitogenic T-cells to theendothelium is an early and very important event in the onset of MS isshown by the finding that anti LFA-1 therapy can completely block theinduction of EAE (Gordon et al., 1995). Additional circumstantialevidence for a stimulatory role of IFNγ in the pathophysiology of MScomes from observations that disease exacerbations are induced by viralupper respiratory infections, known to stimulate the secretion of IFNγby type-2 helper T cells (Panitch, 1994). The proinflammatory role ofIFNγ in autoimmune disease is strengthened by an earlier finding thattreatment of MS patients with hIFNγ resulted in an aggravation of thesymptoms (Panitch et al., 1986). The role of IFNγ as proinflammatorycytokine in autoimmune disorders has been studied in severalexperimentally induced forms of autoimmunity. In experimental neuritis,induced by myelin or antigen-specific T cells in rat, IFNγ clearly actedas pro-inflammatory cytokine and administration of a monoclonal antibodyto IFNγ suppressed the disease (Hartung et al., 1990). In the case ofexperimental autoimmune thyroiditis (EAT) in mice, induced by theinjection of thyroglobulin, treatment of the animals with anti-IFNγ at 4weeks after induction of EAT proved to be beneficial, sincecharacteristic features of EAT such as the lymphocytic infiltrations ofthe thyroid glands and the serum levels of autoantibodies tothyroglobulin, were significantly reduced (Tang et al., 1993).

In the mouse EAE model for MS, where the disease can be induced byinjection of either spinal cord homogenate or myelin basic protein,elevated concentrations of several cytokines, including IFNγ wereobserved both in serum and in the lesions in the CNS (Willenborg et al.,1995). However, administration of anti-IFNγ at the initiation of thedisease, resulted in an exacerbation of the disease (Billiau et al.,1988; Duong et al., 1994; Willenborg et al., 1995). It must be noted,however, that in these experiments the effect of anti-IFNγ wasdetermined at the onset of acute EAE rather than at the time of chronicrelapse of the disease, which in fact is the only relevant situation forMS. Pathologically, typical acute EAE differs substantially from MS inthat prominent inflammation occurs in gray, white and meningealstructures, but demyelisation is scant or absent (Genain et al., 1995b).In order to explain the findings with anti-IFNγ antibodies, the authorssuggest a different action of IFNγ at the systemic level(anti-inflammatory action) compared to the local level (inflammatoryaction) (Billiau et al., 1988), or suggest an early role (within 24hafter immunization) of IFNγ in disease resistance (Duong et al., 1994).Willenborg et al. (1995) conclude that the time of treatment plays acritical role on the outcome and suggest this to be the explanation forconflicting results in different autoimmune processes. Recently,Heremans et al. (1996) described facilitation of spontaneous relapses inchronic relapsing EAE in Biozzi ABH mice by administration of anti-IFNγduring the remission phase. The onset of relapses was delayed whenanimals were treated with IFNγ during the remission phase, results whichare in contradiction to the excacerbation seen in humans who weretreated with hIFNγ.

An experimental EAE model that more closely resembles the disease courseand symptomatology of MS in humans can be found in marmosets. Indeed, inthese animals a chronic relapsing-remitting form of EAE can be inducedwhich is characterized by an initial, acute phase with clinically mildneurological signs, followed by recovery. A late spontaneous relapseoccurs in these animals and chronic lesions resemble active plaques ofchronic MS (Massacesi et al., 1995). This unique model can efficientlybe employed to evaluate a prospective therapy for MS. In this model, acritical role for TNFα in demyelisation is suggested by the observationthat rolipram, a selective inhibitor of the type IV phosphodiesterase,suppressed TNFα secretion and demyelisation (Genain et al., 1995a;Sommer et al., 1995) when administered shortly after immunization, thusinterfering with acute EAE. The effect of anti-IFNγ on acute EAE or ondisease relapse has to our knowledge never been investigated inmarmoset.

Taken together, it is well established that there are a number ofclinical situations in which IFNγ-activity has deleterious effects.Consequently, several potential therapies to neutralize IFNγ-activityhave been proposed. Among the latter proposals are the use of: anti-IFNγantibodies (Ozmen et al., 1995; Bucklin et al., 1994), recombinantanti-IFNγ Fv fragments (EP 0528469 to Billiau & Froyen), bispecificmolecules (WO 94/14467 to Ashkenazi and Ward), drugs such aspentoxifylline (Bienvenu et al., 1995), synthetic polypeptides whichinhibit binding of IFNγ to its receptor (U.S. Pat. No. 5,451,658 toSeelig; U.S. Pat. No. 5,632, 988 to Ingram et al.), Epstein-Barr virusderived proteins (U.S. Pat. No. 5,627,155 to Moore & Kastelein), solubleIFNγ receptors (EP 0393502 to Fountoulakis et al.; U.S. Pat. No.5,578,707 to Novick & Rubinstein) and oligonucleotides which bind toIFNγ (WO95/00529 to Coppola et al.). However, these compounds are facedwith problems such as suboptimal stability, affinity and clearancerates, lack of specificity, efficacy and tissue penetrance, toxic sideeffects and unwanted carrier effects. Indeed, the carrier effect ofantibodies can limit their efficiency to block the target cytokine. Forexample, Montero-Julian et al. (1995) showed that during treatment ofmyeloma patients with anti-IL-6, accumulation of IL-6 in the serum inthe form of monomeric immune complexes occurred, hereby stabilizing thecytokine. Furthermore, it has also been shown that the therapeuticefficacy of a cytokine can be prolonged by the formation ofcytokine/antibody complexes, since the efficacy of recombinant humanIL-2 treatment could be increased by prolonging its in vivo half-life bycomplexing with an anti-IL-2 antibody (Courtney et al., 1994). Thecarrier-effect of anti-cytokine antibodies can be overcome by theconstruction of monovalent scFv fragments, although their low MW(∀30.000) and the associated fast clearance rate, make them lesssuitable candidates for long-term treatment. However, the undesirablecarrier effect can be avoided by the formation of higher immunecomplexes, as such increasing the clearance of the cytokine-antibodycomplexes (Montero-Julian et al., 1995). The use of monoclonalantibodies for diagnostic or therapeutic purposes in vivo is, besidesthe carrier effect, also limited because of their nature (i.e. themajority are murine mAb's and administration of antibodies of mouseorigin inevitably results in a human anti-mouse antibody [HAMA]response), their suboptimal efficacy, stability and affinity and theirlarge molecular size. Proposed solutions to some of these problemsinvolve the use of F(ab′)2, F(ab) and scFv derivatives or of humanizedversions of the parent antibody, either by CDR grafting (Kettleboroughet al., 1991) or by resurfacing of the antibodies (Roguska et al.,1994). Another proposed solution is the development of several modifiedantibodies or antibody constructs by bioengineering or chemical methods.Indeed, some mAb's were made more effective by conjugatingchemotherapeutic drugs and other toxins to the antibodies (Ghetie andVitetta, 1994) or by developing bispecific and/or multivalent antibodyconstructs capable of simultaneously binding several—or two differentepitopes on the same—or different antigens. These antibody constructshave been produced using a variety of methods: a) antibodies ofdifferent specificities or univalent fragments of pepsin-treatedantibodies of different specificities have been chemically linked(Fanger et al., 1992); b) two hybridomas secreting antibodies ofdifferent specificity have been fused and the resulting bispecificantibodies from the mixture of antibodies were subsequently isolated; c)genitically engineered single chain antibodies have been used to producenon-covalently linked bispecific antibodies (e.g. diabodies (Holliger etal., 1993), minibodies (Kostelny et al., 1992) and tetravalentantibodies (Pack et al; 1995; WO 96/13583 to Pack) or covalently-linkedbispecific antibodies (e.g. chelating recombinant antibodies (Kranz etal., 1995), single chain antibodies fused to protein A or Streptavidin(Ito and Kurosawa, 1993; Kipriyanov et al., 1996) and bispecifictetravalent antibodies (EP 0517024 to Bosslet and Deeman). Recently,also trivalent antibody constructs, named triabodies (Kortt et al.,1997), and pentavalent constructs, named peptabodies (Terskikh et al.,1997), have been described. These constructs may have a higher avidityin comparison to bivalent constructs and may be useful for diagnostic ortherapeutic purposes in vivo.

However, and despite the fact that several potential therapies toneutralize IFNγ-activity have been proposed, no prior art existsregarding the production and existence of engineered antibodyconstructs, such as humanized single-chain Fv fragments, diabodies,triabodies, tetravalent antibodies, peptabodies and hexabodies, andruminant-derived antibodies such as sheep antibodies which overcome theabove-indicated problems and which can efficiently be used to treatdiseases wherein interferon-gamma activity is pathogenic.

SUMMARY OF THE INVENTION

It is clear from the prior art as cited above that problems such assuboptimal stability, affinity, clearance rate, specificity, efficacy,and an unwanted carrier effect and HAMA response hamper the successfulusage of several therapeutics which, potentially, could neutralize theactivity of IFNγ. Also suggested solutions to overcome some of theseproblems did not result in the development of effective products. Thus,unpredictable and unknown factors still appear to determine the successof these biologicals. Despite these unknown factors, the presentinventors have been able to design and develop useful constructs whicheffectively neutralize IFNγ-activity. Indeed, the constructs have all asurprisingly high affinity for IFNγ, they do not provoke a HAMA orrelated response, and they do not result in a carrier effect. Inaddition, some of the constructs pass the blood brain barrier, whereasothers have a very good clearance rate. Therefore, the present inventionaims at providing a molecule which binds and neutralizesinterferon-gamma and which is chosen from the group consisting of:

a scFv comprising the humanized variable domain of the monoclonalantibody D9D10

a chimeric antibody comprising the humanized variable domain of themonoclonal antibody D9D10

a diabody comprising the humanized variable domain of the monoclonalantibody D9D10

a multivalent antibody

a ruminant antibody.

The present invention further aims at providing a multivalent antibodychosen from the group consisting of triabodies, tetravalent antibodies,peptabodies and hexabodies.

The present invention also aims at providing a triabody, tetravalentantibody, peptabody and hexabody which comprise 3, 4, 5 and 6 variabledomains, respectively, of different anti-interferon-gamma antibodies.

The present invention further aims at providing a triabody as describedabove which comprises 3 identical variable domains of ananti-interferon-gamma antibody. A preferred variable domain used in thelatter constructs is derived from the mouse anti-interferon-gammaantibody D9D10 which is described by Sandvig et al. (1987) and Froyen etal. (1993) or from the sheep anti-interferon-gamma antibody described inthe present application. Therefore, the present invention aims atproviding a triabody as described above which comprises 3 identicalD9D10 scFv's, 3 identical humanized D9D10 scFv's, 3 identicalsheep-derived anti-interferon-gamma scFv's or 3 identical humanizedsheep-derived anti-interferon-gamma scFv's.

The present invention further aims at providing a tetravalent antibody(called MoTAb I) as described above which comprises 4 identical domainsof an anti-interferon-gamma antibody. More specifically, the presentinvention aims at providing a tetravalent antibody as described abovewhich comprises either 4 identical D9D10 scFv's or 4 identicalsheep-derived anti-interferon-gamma scFv's in the format of a homodimerof 2 identical molecules, each containing 2 D9D10 scFv's or 2 humanizedD9D10 scFv's or 2 sheep-derived anti-interferon-gamma scFv's or 2humanized sheep-derived anti-interferon-gamma scFv's, and a dimerizationdomain, or, a full-size humanized D9D10 antibody or sheep-derivedanti-interferon-gamma antibody to which 2 humanized D9D10 scFv's or 2humanized sheep-derived anti-interferon-gamma scFv's, respectively, areattached at the carboxyterminus (called MoTAb II) (see FIG. 1).

The present invention further aims at providing a peptabody and hexabodyas described above which comprise 5 and 6 identical variable domains ofan anti-interferon-gamma antibody, respectively. A preferred variabledomain used in the latter constructs is derived from the mouseanti-interferon-gamma antibody D9D10 which is described above or fromthe sheep anti-interferon-gamma antibody described in the presentapplication. Therefore, the present invention aims at providing apeptabody and hexabody as described above which comprises 5 or 6identical D9D10 scFv's, 5 or 6 identical humanized D9D10 scFv's, 5 or 6identical sheep-derived anti-interferon-gamma scFv's, or, 5 or 6identical humanized sheep-derived anti-interferon-gamma scFv's,respectively.

The present invention further aims at providing a molecule as describedabove, wherein said ruminant antibody is a sheep antibody.

The present invention also aims at providing a molecule as describedabove, wherein said sheep antibody is a monoclonal antibody.Furthermore, the present invention aims at providing a humanizedantibody, a single-chain fragment or any other fragment which is derivedfrom said monoclonal antibody and which has largely retained thespecificity of said monoclonal antibody.

Moreover, the present invention aims at providing methods for producingthe above-described molecules.

The present invention further aims at providing a pharmaceuticalcomposition comprising a molecule as described above, or a mixture ofsaid molecules, in a pharmaceutically acceptable excipient.

The present invention also aims at providing a molecule or a compositionas described above for use as a medicament.

Furthermore, the present invention aims at providing a molecule or acomposition as described above for preventing or treating septic shock,cachexia, immune diseases such as multiple sclerosis and Crohn's diseaseand skin disorders such as bullous, inflammatory and neoplasticdermatosis.

Finally, the present invention aims at providing a molecule as describedabove for determining interferon gamma levels in a sample.

All the aims of the present invention are considered to have been met bythe embodiments as set out below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 schematically shows 2 different tetravalent antibody constructs(MoTAB I and MotabII). MoTAb I represents a molecule which consists of 4identical scFv's in the format of a homodimer of 2 identical molecules,each containing 2 scFv's. MoTAb II represents a full-size antibodymolecule to which 2 scFv's with the same specificity are attached at thecarboxyterminus. Optionally, these constructs contain apurification/detection tag.

See also further Example 4.

FIG. 2 shows the coding (SEQ ID NO 1) and amino acid sequence (SEQ ID NO2) of humanized D9D10 scFv (containing a C-terminal 6-histidine tag(bold)). CDR regions are underlined. Mutations (murine->human) are boldand underlined. The N-terminal pelB signal sequence is put in bold.

FIGS. 3 and 4 shows the binding of different concentrations of murinescFvD9D10 (FIG. 3) and humanized scFvD9D10 (FIG. 4) to human IFNγ. HumanIFNγ is immobilized indirectly to the CM5 sensorchip via the murine D9D10 full size antibody as described in example 1. Association rateconstants derived from these binding curves are shown. Dissociation rateconstants could not be measured accurately as dissociation is hardlydetectable (<5×10⁻⁴ s⁻¹) in this experimental setup.

FIG. 5 shows a schematic representation of the mammalian expressionplasmid pEE12hD9D10 used for expression of humanized D9D10 wholeantibody in (1) COS cells (2) stable recombinant Ns0 cell lines.

Major Plasmid Building Blocks:

prokaryotic sequences for plasmid DNA preparation in E.coli (ori ofreplication and amp^(R) ampicilline resistance expression unit)

SV40 origin of replication (part of SV40E, SV40 early promoter) allowingtransient expression in SV40 permissive, T-antigen producing cell lines(e.g. COS)

human Cytomegalovirus major immediate early promoter/enhancer(hCMVprom+intron) controlled expression casette for hD9D10 heavy chainprotein (hD9D10-H)

human Cytomegalovirus major immediate early promoter/enhancer(hCMVprom+intron) controlled expression casette for hD9D10 light chainprotein (hD9D10-L)

SV40 early promoter (SV40E) controlled glutamine synthetase cDNA (GS)expression unit for selection/amplification

polyA=SV40 early region poly-adenylation signal intron+polyA=SV40t-antigen intron +SV40 early region poly-adenylation signal

FIG. 6 shows a schematic representation of the mammalian expressionplasmid pEE14hD9D10 used for expression of humanized D9D10 wholeantibody in (1) COS cells (2) stable recombinant CHO-K1 cell lines.

Major Plasmid Building Blocks:

prokaryotic sequences for plasmid DNA preparation in E. coli (ori ofreplication and amp^(R) ampicilline resistance expression unit)

SV40 origin of replication (part of SV40E, SV40 early promoter) allowingtransient expression in SV40 permissive, T-antigen producing cell lines(e.g. COS)

human Cytomegalovirus major immediate early promoter/enhancer(hCMVprom+intron) controlled expression casette for hD9D10 heavy chainprotein (hD9D10-H)

human Cytomegalovirus major immediate early promoter/enhancer(hCMVprom+intron) controlled expression casette for hD9D10 light chainprotein (hD9D10-L)

SV40 late promoter (SV40L) controlled glutamine synthetase mini gene(GS+intron) expression unit for selection/amplification

polyA=SV40 early region poly-adenylation signal

intron+polyA=SV40 t-antigen intron+SV40 early region poly-adenylationsignal

FIG. 7 shows the cDNA sequence encoding the humanized D9D10 heavy chainfusion protein.

bp 1-60: D9D10 Kappa-light chain signal sequence

bp 61-411: humanized D9D10 heavy chain variable domain

bp 412-1401: human IgG1 heavy chain constant domain(C_(H)1-Hinge-C_(H)2-C_(H)3)

bp 1402-1404: leu codon added by PCR cloning strategy (SEQ ID NO 66)

FIG. 8 shows the cDNA sequence encoding the humanized D9D10 and MoTAbIIlight chain fusion protein.

bp 1-60: D9D10 Kappa-light chain signal sequence

bp 61-381: humanized D9D10 light chain variable domain

bp 382-699: human kappa light chain constant domain (SEQ ID NO 68)

FIG. 9 shows the amino acid sequence of the humanized D9D10 heavy chainfusion protein.

Aa 1-20: D9D10 light chain signal sequence

Aa 21-137 : humanized heavy chain variable domain of D9D10

Aa138-467: human IgG1 heavy chain constantdomain(C_(H)1-hinge-C_(H)2-C_(H)3)

Aa 468: leu added by PCR cloning strategy

Aa 351: pro was mutated to ser: inactivation C1q complement binding

Number of residues: 468.

Molecular weight (MW): 51413 (SEQ ID NO 67)

FIG. 10 shows the amino acid sequence of the humanized D9D10 and MoTAbIIlight chain fusion protein.

Aa 1-20: D9D10 light chain signal sequence

Aa 21-127: humanized light chain variable domain of D9D10

Aa 128-233: human kappa light chain constant domain

Number of residues: 233.

Molecular weight (MW): 25582 (SEQ ID NO 69)

FIG. 11 shows the binding in ELISA of different concentrations ofhumanized D9D10 and humanized D9D10 MoTabII (=different dilutions ofcrude COS supernatant containing humanized D9D10 or humanized D9D10MotabII) to immobilized human IFN. The assay is performed as describedin example 2.

FIG. 12 shows the interaction of humanized D9D10 (=crude COS supernatantcontaining humanized D9D10) with IFN using SPR analysis. The assay isperformed as described in example 2.

FIG. 13 shows the binding in ELISA of different concentrations ofpurified humanized D9D10 and MoTabII to immobilized human IFNγ. Theassay is performed as described in example 2.

FIG. 14 shows a schematic representation of the expressionplasmidpMoTAbIH6 used for the expression of MoTAbI in E.coli.

FIG. 15 shows the cDNA sequence of MoTAbI

bp 1-351: V_(H) D9D10

bp 352-396: (G₄S)₃ linker

bp 397-717: V_(L) D9D10

bp 718-750: human IgG3 upper hinge

bp 751-855: helix-turn-helix dimerisation domain

bp 856-888: human IgG3 upper hinge

bp 889-1239: V_(H)D9D10

bp 1240-1284:(G₄S)₃ linker

bp 1285-1605: V_(L) D9D10

bp 1606-1623: His6 tag (SEQ ID NO 84)

FIG. 16 shows the AA sequence of MoTAbI

aa 1-117: V_(H) D9D10

aa 118-132: (G₄S)₃ linker

aa 133-239: V_(L) D9D10

aa 240-250: human IgG3 upper hinge

aa 251-285: helix-turn-helix dimerisation domain

aa 286-296: human IgG3 upper hinge

aa 297-413: V_(H) D9D10

aa 414-428: (G₄S)₃ linker

aa 429-525: V_(L) D9D10

aa 526-531: His6 tag (SEQ ID NO 85)

FIG. 17 shows a schematic representation of the mammalian expressionplasmid pEE12MoTAbII used for expression of D9D10 MoTAbII recombinantantibody in (1) COS cells (2) stable recombinant Ns0 cell lines.

Major Plasmid Building Blocks:

prokaryotic sequences for plasmid DNA preparation in E.coli (ori ofreplication and amp^(R) ampicilline resistance expression unit)

SV40 origin of replication (part of SV40E, SV40 early promoter) allowingtransient expression in SV40 permissive, T-antigen producing cell lines(e.g. COS)

human Cytomegalovirus major immediate early promoter/enhancer(hCMVprom+intron) controlled expression casette for D9D10MoTAbII heavychain protein (MoTAbII-H)

human Cytomegalovirus major immediate early promoter/enhancer(hCMVprom+intron) controlled expression casette for D9D10MoTAbII lightchain protein (MoTAbII-L)

SV40 early promoter (SV40E) controlled glutamine synthetase cDNA (GS)expression unit for selection/amplification

polyA=SV40 early region poly-adenylation signal

intron+polyA=SV40 t-antigen intron+SV40 early region poly-adenylationsignal

FIG. 18 shows a schematic representation of the mammalian expressionplasmid pEE14MoTAbII used for expression of D9D10MoTAbII recombinantantibody in (1) COS cells (2) stable recombinant CHO-K1 cell lines.

Major Plasmid Building Blocks:

prokaryotic sequences for plasmid DNA preparation in E.coli (ori ofreplication and amp^(R) ampicilline resistance expression unit)

SV40 origin of replication (part of SV40E, SV40 early promoter) allowingtransient expression in SV40 permissive, T-antigen producing cell lines(e.g. COS)

human Cytomegalovirus major immediate early promoter/enhancer(hCMVprom+intron) controlled expression casette for D9D10MoTAbII heavychain protein (MoTAbII-H)

human Cytomegalovirus major immediate early promoter/enhancer(hCMVprom+intron) controlled expression casette for D9D10MoTAbII lightchain protein (MoTAbII-L)

SV40 late promoter (SV40L) controlled glutamine synthetase mini gene(GS+intron) expression unit for selection/amplification

polyA=SV40 early region poly-adenylation signal

intron+polyA=SV40 t-antigen intron+SV40 early region poly-adenylationsignal

FIG. 19 shows the cDNA sequence encoding the MoTABII fusion protein

bp 1-60: D9D10 Kappa-light chain signal sequence

bp 61-411: humanized D9D10 heavy chain variable domain

bp 412-1401: human IgG1 heavy chain constant domain(C_(H)1-Hinge-C_(H)2-C_(H)3)

bp 1402-1404: leu codon added by PCR cloning strategy

bp 1405-1416 : gly(3)-ser codon

bp 1417-2133: humanized D9D10 ScFv (SEQ ID NO 8: 9)

FIG. 20 shows the amino acid sequence of MoTABII fusion protein

Aa 1-20: mouse D9D10 light chain signal sequence

Aa 21-137: humanized heavy chain variable domain of D9D10

Aa 138-467: human IgG1 heavy chain constantdomain(C_(H)1-hinge-C_(H)2-C_(H)3)

Aa 351: pro mutated to ser: inactivation C1q complement binding

Aa 468: leu added by cloning strategy

Aa 469-472: gly(3)-ser linker

Aa 473-711: humanized D9D10 ScFv (V_(H)473-490/gly-serlinker/V_(L)605-711) (SEQ ID NO 90)

FIG. 21 shows the interaction of MoTAbII (=crude COS supernatantcontaining MoTAbII) with IFNγ using SPR analysis. The assay is performedas described in example 4.

FIG. 22 shows the amino acid sequence of the D9D10 L10 diabody

aa 1-117: V_(H) D9D10

aa 118-127: (G₄S)₂ linker

aa 128-234: V_(L) D9D10

aa 235-240: His6-tag (SEQ ID NO 91)

FIG. 23 shows the coding sequence of the D9D10 L10 diabody

bp 1-351: V_(H) D9D10

bp 352-381: (G₄S)₂ linker

bp 382-702: V_(L) D9D10 (SEQ ID NO 92)

FIG. 24 shows the amino acid sequence of the D9D10 L5 diabody

aa 1-117: V_(H) D9D10

aa 118-122: G₄S linker

aa 123-229: V_(L) D9D10

aa 230-235: His6-tag 5SEQ ID NO 93)

FIG. 25 shows the coding sequence of the D9D10 L5 diabody

bp 1-351: V_(H) D9D10

bp 352-366: G₄S linker

bp 367-687: V_(L) D9D10 (SEQ ID NO 94)

FIG. 26 shows the interaction of humanized L5 D9D10 diabody (=crudelysate from E. coli) with IFN using SPR analysis. The assay is performedas described in example 5.

FIG. 27 shows the coding sequence of the D9D10 triabody

bp 1-351: V_(H) D9D10

bp 352-672: V_(L) D9D10 (SEQ ID NO 101)

FIG. 28 shows the amino acid sequence of the D9D10 L0 triabody

aa 1-117: V_(H) D9D10

aa118-224:V_(L) D9D10

aa 225-230: His6-tag (SEQ ID NO 102)

FIG. 29 shows the interaction of humanized L0 D9D10 triabody (=crudelysate from E. coli) with IFNγ using SPR analysis. The assay isperformed as described in example 6.

FIG. 30 shows the neutralization of IFN-gamma-induced MHC class IIupregulation on human primary keratinocytes by D9D10 or D9D10 scFv.Human keratinocytes were cultured for 24 h with or without (not shown)100 U/ml huIFN-gamma in the absence or the presence of D9D10 (2 μg/ml).Resting human keratinocytes do not express MHC class II. IFN-gammainduces expression of MHC class II in the keratinocytes and D9D10 (upperpanel) or scFv D9D10 (lower panel) inhibit this IFN-gamma-induced MHCclass II expression. See also further Example 7.1.

FIG. 31 shows the neutralization of IFN-gamma-induced MHC class IIupregulation on human primary keratinocytes by crude COS supernatantcontaining either humanized D9D10 or MoTAbII. The experiment wasperformed as described in FIG. 30

thin line:

human keratinocytes treated with human IFNγ

bold line:

A: human keratinocytes not treated with human IFNγ

B: effect of 400 ng/ml murine D9D10

C: effect of humanized D9D10 (crude COS supernatant)

D: effect of MoTAbII (crude COS supernatant)

FIG. 32 shows the effect of the anti-IFN-gamma antibody F3 and scFvF3 onthe survival of mice in which the lethal shock syndrome called“Shwartzman reaction” is induced. See also further Example 7.3.

FIG. 33 shows the effect of the anti-IFN-gamma antibody F3 and scFvF3 onbody weight of mice exhibiting IFN-gamma induced cachexia. Mortality(number of dead mice/total number of mice) is shown between brackets andthe symbol “+”. See also further Example 7.4.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention described herein draws on previously published work andpending patent applications. By way of example, such work consists ofscientific papers, patents or pending patent applications. All of thesepublications and applications, cited previously or below are herebyincorporated by reference.

The present invention is based on the finding that a molecule whichbinds and neutralizes human interferon-gamma and which is chosen fromthe group consisting of:

a scFv comprising the humanized variable domain of the monoclonalantibody D9D10

a chimeric antibody comprising the humanized variable domain of themonoclonal antibody D9D10

a diabody comprising the humanized variable domain of the monoclonalantibody D9D10

a multivalent antibody

a ruminant antibody is useful to treat diseases where IFNγ activity ispathogenic.

As used herein the terms “molecule which binds and neutralizes IFNγ”refer to a molecule which recognizes and binds any particular epitope ofIFNγ resulting in the neutralization of any bioactivity of IFNγ.Particular epitopes of IFNγ relate to the so-called E2 epitoperecognized and bound by the mAb D9D10, the so-called E1 epitope (Kwok etal., 1993) or any other epitope. IFNγ specifically relates to human IFNγbut may also relate to non-human primate, mouse, rat, sheep, goat,camel, cow, llama or any other IFNγ. Furthermore, the term “bioactivityof IFNγ” relates to the antiviral activity (Billiau, 1996), theinduction of the expression of MHC-class-II molecules by macrophages andother cell types (Steinman et al., 1980), the stimulation of theproduction of inflammatory mediators such as TNFα, IL-1 and NO (Lorsbachet al., 1993), the induction of the expression of adhesion moleculessuch as ICAM-1 (Dustin et al., 1988) and of important costimulators suchas the B7 molecules on professional antigen presenting cells (Freedmanet al., 1991), the induction of macrophages to become tumoricidal (Paceet al., 1983), the induction of Ig isotype switching (Snapper and Paul,1987), any pathological and/or clinical activity during diseases whereIFNγ is pathogenic (Billiau, 1996) or any other known bioactivity ofIFNγ. In this regard, it should be clear that any assay systemdemonstrating the IFNγ-neutralizing capacity of a molecule, such as theones described by Novelli et al. (1991), Lewis (1995) and Turano et al.(1992) can be used. Some of these assays are also described in thesubsection Evaluation of anti-IFNγ neutralizing molecules in theExamples section of the present application (see further). It should benoted that the molecules which bind and neutralize IFN- γ as describedabove neutralize at least one bioactivity, but not necessarily allbioactivities, of IFN-γ.

The present invention further relates to a scFv comprising the humanizedvariable domain of the monoclonal antibody D9D10. As used herein, theterm single-chain Fv, also termed single-chain antibody, refers toengineered antibody constructs prepared by isolating the binding domains(both heavy and light chain) of a binding antibody, and supplying alinking moiety which permits preservation of the binding function. Thisforms, in essence, a radically abbreviated antibody, having only thevariable domain necessary for binding the antigen. Determination andconstruction of single chain antibodies are described in U.S. Pat. No.4,946,778 to Ladner et al. and in the Examples section of the presentapplication (see further). The term “humanized” means that at least aportion of the framework regions of an immunoglobulin or engineeredantibody construct is derived from human immunoglobulin sequences. Itshould be clear that any method to humanize antibodies or antibodyconstructs, as for example by variable domain resurfacing as describedby Roguska et al. (1994) or CDR grafting or reshaping as reviewed byHurle and Gross (1994), can be used. The humanization of the scFvcomprising the variable domain of the monoclonal antibody D9D10 isdescribed further in the Examples section of the present application.The monoclonal antibody D9D10 was prepared essentially as described bySandvig et al. (1987) and Froyen et al. (1993). It should also be notedthat the process of humanization of an antibody or antibody construct isregularly accompanied by a significant loss in binding affinity of thisantibody or antibody construct (Kettleborough et al., 1991; Park et al.,1996 and Mateo et al., 1997). In contrast, and surprisingly, theconstructs humanized by the present inventors were not characterized bya significant loss in binding affinity in comparison to theirnon-humanized counterparts.

The present invention also relates to a chimeric antibody comprising thehumanized variable domain of the monoclonal antibody D9D10. The term“chimeric antibody” refers to an engineered antibody constructcomprising variable domains of one species (such as mouse, rat, goat,sheep, cow, llama or camel variable domains), which may be humanized ornot, and constant domains of another species (such as non-human primateor human constant domains) (for review see Hurle and Gross (1994)). Itshould be clear that any method known in the art to develop chimericantibodies or antibody constructs can be used. The generation of achimeric antibody comprising the humanized variable domain of themonoclonal antibody D9D10 is described further in the Examples sectionof the present application.

The present invention also concerns a diabody comprising the humanizedvariable domain of the monoclonal antibody D9D10. The term “diabody”relates to two non-covalently-linked scFv's, which then form a so-calleddiabody, as described in detail by Holliger et al. (1993) and reviewedby Poljak (1994). It should be clear that any method to generatediabodies, as for example described by Holliger et al. (1993), Poljak(1994) and Zhu et al. (1996), can be used. The generation of diabodiescomprising the variable domain of the monoclonal antibody D9D10 isdescribed further in the Examples section of the present application.

It should also be clear that the scFv's, chimeric antibodies anddiabodies described above are not limited to comprise the variabledomain of the monoclonal antibody D9D10 but may also comprise variabledomains of other anti-IFNγ antibodies, such as the sheep anti-IFNγantibody described further in the present application, which efficientlyneutralize the bioactivity of IFNγ.

Furthermore, the diabodies described above may also comprise two scFv'sof different specificities. For example, the latter diabodies maysimultaneously neutralize IFN on the one hand and may target anothermolecule, such as TNF-α, IL-1, IL-2, B7.1 or CD80, B7.2 or CD86, IL-12,IL-4, IL-10, CD40, CD40L, IL-6, tumour growth factor-beta (TGF-β),transferrin receptor, insulin receptor and prostaglandin E2 or any othermolecule, on the other hand.

The present invention also concerns multivalent antibodies which bindand neutralize IFNγ. As used herein, the term multivalent antibodyrefers to any IFNγ-binding and IFNγ-neutralizing molecule which has morethan two IFNγ-binding regions. Examples of such multivalent antibodiesare triabodies, tetravalent antibodies, peptabodies and hexabodies whichbind and neutralize IFNγ and which have three, four, five and sixIFNγ-binding regions, respectively.

The present invention thus relates, as indicated above, to triabodieswhich bind and neutralize IFNγ. As used herein, the term “triabody”relates to trivalent constructs comprising 3 scFv's, and thus comprising3 variable domains, as described by Kortt et al. (1997) and Iliades etal. (1997). A method to generate triabodies is described by Kortt et al.(1997) and the generation of triabodies comprising the variable domainof the monoclonal antibody D9D10 is described further in the Examplessection of the present application. It should be noted that thetriabodies of the present invention may comprise: 3 variable domains of3 different anti-IFNγ Ab's (i.e. 3 anti-IFNγ Ab's which recognize andbind a different epitope on IFNγ [see also above]), 3 variable domainsof 3 identical anti-IFNγ Ab's such as 3 variable domains of D9D10 or 3variable domains of humanized D9D10 or 3 variable domains of sheepanti-IFNγ Ab's or 3 humanized variable domains of sheep anti-IFNγ Ab's,1 or 2 variable domain(s) of anti-IFNγ Ab's in combination with 2 or 1variable domain(s) of an Ab which binds to any other molecule than IFNγ,respectively. Examples of such other molecules comprise TNFαa, IL-1,IL-2, B7.1 or CD80, B7.2 or CD86, IL-12, IL-4, IL-10, CD40, CD40L, IL-6,tumour growth factor-beta (TGF-β), transferrin receptor, insulinreceptor and prostaglandin E2.

The present invention further relates to tetravalent antibodies whichbind and neutralize IFNγ. As used herein, the term “tetravalentantibody” refers to engineered antibody constructs comprising 4antigen-binding regions as described by Pack et al. (1995) and Coloma &Morrison (1997). Methods to generate these tetravalent antibodyconstructs are also described by the latter authors. The generation ofthe following 2 different tetravalent antibodies comprising the variabledomain of the monoclonal antibody D9D10 are described further in theExamples section of the present application: MoTabI which consists of 4identical humanized D9D10 scFv's in the format of a homodimer of twoidentical molecules each containing two D9D10 scFv's which are linkedtogether using a dimerization domain; the latter domain also drives thehomodimerization of the molecule, and, MoTab II which consists of afull-size humanized D9D10 molecule to which two humanized D9D10 scFv'sare attached at the carboxyterminus (CH3-domain). It should be notedthat the tetravalent antibodies of the present invention may comprise: 4variable domains of 4 different anti-IFNγ Ab's (i.e. anti-IFNγ Ab'swhich recognize and bind to a different epitope on IFNγ), 4 variabledomains of 4 identical anti-IFNγ Ab's such as 4 variable domains ofD9D10 or 4 variable domains of humanized D9D10 or 4 variable domains ofsheep anti-IFNγ Ab's or 4 humanized variable domains of sheep anti-IFNγAb's, 2 variable domain(s) of one anti-IFNγ Ab in combination with 2variable domain(s) of another anti-IFNγ Ab, 2 variable domain(s) ofanti-IFNγ Ab's in combination with 2 variable domain(s) which binds toany other molecule than IFNγ. Examples of such other molecules compriseTNFα, IL-1, IL-2, B7.1 or CD80, B7.2 or CD86, IL-12, IL-4, IL-10, CD40,CD40L, IL-6, TGF-β, transferrin receptor, insulin receptor andprostaglandin E2.

Furthermore, the term “dimerization domain” of MoTab I refers to anymolecule known in the art which is capable of coupling the two identicalmolecules. Examples of such domains are the leucine zipper domain (deKruif & Logtenberg, 1996), the helix-turn-helix motif described by Packet al. (1993), the max-interacting proteins and related molecules asdescribed in U.S. Pat. No. 5,512,473 to Brent & Zervos and thepolyglutamic acid-polylysine domains as described in U.S. Pat. No.5,582,996 to Curtis.

The present invention thus relates, as indicated above, to peptabodiesand hexabodies which bind and neutralize IFNγ. As used herein, the term“peptabodies” relates to pentavalent constructs as described in detailby Terskikh et al. (1997). The term “hexabodies” relates to hexavalentconstructs which are similar to the pentavalent constructs as describedin detail by Terskikh et al. (1997) but wherein the pentamerizationdomain is replaced by any hexamerization domain known in the art. Amethod to generate peptabodies is also described by Terskikh et al.(1997) and a method to generate hexabodies can be derived from thedescription by the latter authors. It should be noted that thepeptabodies and hexabodies of the present invention may comprise: 5(relating to the peptabodies) or 6 (relating to the hexabodies) variabledomains of 5 or 6 different anti-IFNγ Ab's (i.e. 5 or 6 anti-IFNγ Ab'swhich recognize and bind a different epitope on IFNγ [see also above]),5 or 6 variable domains of identical anti-IFNγ Ab's such as 5 or 6variable domains of D9D10, or, 5 or 6 variable domains of humanizedD9D10, or, 5 or 6 variable domains of sheep anti-IFNγ Ab's, or, 5 or 6humanized variable domains of sheep anti-IFNγ Ab's, less than 5 or 6variable domain(s) of any anti-IFNγ Ab's in combination with less than 5or 6 variable domain(s) of an Ab which binds to any other molecule thanIFNγ, respectively. Examples of such other molecules comprise TNFα,IL-1, IL-2, B7.1 or CD80, B7.2 or CD86, IL-7.2, IL-4, IL-10, CD40,CD40L, IL-6, TGF-β, transferrin receptor, insulin receptor andprostaglandin E2.

The present in invention further relates to ruminant antibodies whichbind and neutralize IFNγ. The term “ruminant” relates to animalsbelonging to the suborder Ruminantia of even-toed hoofed mammals (assheep, goats, cows, giraffes, deer, llama, vicunas and camels) that chewthe cud and have a complex 3- or 4-chambered stomach.

More specifically, the present invention relates to sheep antibodieswhich bind and neutralize IFNγ. The term “sheep” relates to any ofnumerous ruminant mammals belonging to the genus Ovis. The generation ofsheep anti-IFNγ antibodies is described in the Examples section of thepresent application. The present invention also relates to sheepmonoclonal antibodies. As used herein, the term “monoclonal antibody”refers to an antibody composition having a homogeneous antibodypopulation. The term is not limited regarding the species or source ofthe antibody, nor is it intended to be limited by the manner in which itis made. Indeed, the monoclonal sheep antibodies of the presentinvention can be generated by any method known in the art. It should benoted that also humanized antibodies, scFv's or any other fragmentthereof which has largely retained the specificity of said sheepantibody or sheep monoclonal antibody are covered by the presentinvention. As used herein, the term “fragment” refers to F(ab), F(ab′)2,Fv, and other fragments which retain the antigen binding function andspecificity of the parent antibody. It should also be understood thatthe variable domains of the sheep anti-IFNγ (monoclonal) antibodies orscFv of the sheep anti-IFNγ (monoclonal) antibodies may be part of thechimeric antibodies, diabodies, triabodies, tetravalent antibodies,peptabodies and hexabodies as described above.

The present invention further relates to scFv's, chimeric antibodies,diabodies, triabodies, tetravalent antibodies, peptabodies, hexabodiesand sheep antibodies which bind and neutralize IFNγ and which areproduced by the methods as described above and in the Examples sectionof the present application.

The present invention further relates to a composition comprising scFv'sand/or chimeric antibodies and/or diabodies and/or triabodies and/ortetravalent antibodies and/or peptabodies and/or hexabodies and/or sheepantibodies which bind and neutralize IFNγ in a pharmaceuticallyacceptable excipient, possibly in combination with other drugs or otherantibodies, antibody derivatives or constructs for use as a medicamentto prevent or treat septic shock, cachexia, immune diseases such asmultiple sclerosis and Crohn's disease and skin disorders such asbullous, inflammatory and neoplastic dermatoses. Examples of such otherdrugs or other antibodies, antibody derivatives or constructs are, withregard to septic shock: an isotonic crystalloid solution such as saline,dopamine, adrenaline and antibiotics; with regard to cachexia:anti-TNF-alpha antibodies; with regard to multiple sclerosis: ACTH andcorticosteroids, interferon beta-1b (Betaseron), interferon beta-1a(Avonex), immunosuppressive drugs such as azathioprine, methotrexate,cyclophosphamide, cyclosporin A and cladribine (2-CdA), copolymer 1(composed of 4 amino acids common to myelin basic proteins), myelinantigens, roquinimex A, the mAb CAMPATH-1H and potassium channelblockers; with regard to Crohn's disease: sulfasalazine,corticosteroids, 6 mercaptopurine/azathioprine and cyclosporin A; withregard to psoriasis: cyclosporin A, methotrexate, calcipotriene(Dovonex), zidovudine (Retrovir), histamine2 receptor antagonists suchas ranitidine (Zantac) and cimetidine (Tagamet), propylthiouracil,acitretin (Soriatane), fumaric acid, vitamin D derivates, tazarotene(Tazorac), IL-2 fusion toxin, tacrolimus (Prograf), CTLA4Ig, anti-CD4mAb's and T-cell receptor peptide vaccines. It should also be clear thatany possible mixture of the above-indicated IFN-γ-binding molecules maybe part of the above-indicated pharmaceutical composition.

As used herein, the term “composition” refers to any compositioncomprising as an active ingredient scFv's and/or chimeric antibodiesand/or diabodies and/or triabodies and/or tetravalent antibodies and/orpeptabodies and/or hexabodies and/or sheep antibodies which bind andneutralize IFNγ according to the present invention possibly in thepresence of suitable excipients known to the skilled man. The scFv'sand/or chimeric antibodies and/or diabodies and/or triabodies and/ortetravalent antibodies and/or peptabodies and/or hexabodies and/or sheepantibodies which bind and neutralize IFNγ of the invention may thus beadministered in the form of any suitable composition as detailed belowby any suitable method of administration within the knowledge of askilled man. The preferred route of administration is parenterally. Inparenteral administration, the compositions of this invention will beformulated in a unit dosage injectable form such as a solution,suspension or emulsion, in association with a pharmaceuticallyacceptable excipient. Such excipients are inherently nontoxic andnontherapeutic. Examples of such excipients are saline, Ringer'ssolution, dextrose solution and Hank's solution. Nonaqueous excipientssuch as fixed oils and ethyl oleate may also be used. A preferredexcipient is 5% dextrose in saline. The excipient may contain minoramounts of additives such as substances that enhance isotonicity andchemical stability, including buffers and preservatives.

The scFv's and/or chimeric antibodies and/or diabodies and/or triabodiesand/or tetravalent antibodies and/or peptabodies and/or hexabodiesand/or sheep antibodies which bind and neutralize IFNγ of the inventionare administered at a concentration that is therapeutically effective totreat or prevent septic shock, cachexia, immune diseases such asmultiple sclerosis and Crohn's disease and skin disorders such asbullous, inflammatory and neoplastic dermatoses. The dosage and mode ofadministration will depend on the individual. Generally, thecompositions are administered so that the scFv's and/or chimericantibodies and/or diabodies and/or triabodies and/or tetravalentantibodies and/or peptabodies and/or hexabodies and/or sheep antibodieswhich bind and neutralize IFNγ are given at a dose between 1 μg/kg and10 mg/kg, more preferably between 10 μg/kg and 5 mg/kg, most preferablybetween 0.1 and 2 mg/kg for each IFN-γ-binding molecule. Preferably,they are given as a bolus dose. Continuous short time infusion (during30 minutes) may also be used. If so, the scFv's and/or chimericantibodies and/or diabodies and/or triabodies and/or tetravalentantibodies and/or peptabodies and/or hexabodies and/or sheep antibodieswhich bind and neutralize IFNγ or compositions comprising the same maybe infused at a dose between 5 and 20 μg/kg/minute, more preferablybetween 7 and 15 μg/kg/minute (for each IFN-γ-binding molecule).

According to the specific case, the “therapeutically effective amount”of a scFv's and/or chimeric antibodies and/or diabodies and/ortriabodies and/or tetravalent antibodies and/or peptabodies and/orhexabodies and/or sheep antibodies which bind and neutralize IFNγ neededshould be determined as being the amount sufficient to cure the patientin need of treatment or at least to partially arrest the disease and itscomplications. Amounts effective for such use will depend on theseverity of the disease and the general state of the patient's health.Single or multiple administrations may be required depending on thedosage and frequency as required and tolerated by the patient.

The present invention further relates to scFv's and/or chimericantibodies and/or diabodies and/or triabodies and/or tetravalentantibodies and/or peptabodies and/or hexabodies and/or sheep antibodieswhich bind and neutralize IFNγ for determining IFNγ levels in abiological sample, comprising:

1) contacting the biological sample to be analysed for the presence ofIFNγ with a scFv and/or chimeric antibody and/or diabody and/or triabodyand/or tetravalent antibody and/or peptabodies and/or hexabodies and/orsheep antibody as defined above,

2) detecting the immunological complex formed between IFNγ and said scFvand/or chimeric antibody and/or diabody and/or triabody and/ortetravalent antibody and/or peptabodies and/or hexabodies and/or sheepantibody.

As used herein, the term “a method to detect” refers to any immunoassayknown in the art such as assays which utilize biotin and avidin orstreptavidin, ELISA's and immunoprecipitation, immunohistochemicaltechniques and agglutination assays. A detailed description of theseassays is given in WO 96/13590 to Maertens & Stuyver. Theimmunohistochemical detection of IFNγ in cryosections of spinal cord andbrain of non-human primates suffering from experimental autoimmuneencephalomyelitis is described in detail in the Examples section of thepresent application. The term “biological sample” relates to anypossible sample taken from a mammal including humans, such as blood(which also encompasses serum and plasma samples), sputum, cerebrospinalfluid, urine, lymph or any possible histological section, wherein IFNγmight be present.

The present invention will now be illustrated by reference to thefollowing examples which set forth particularly advantageousembodiments. However, it should be noted that these embodiments areillustrative and are not to be construed as restricting the invention inany way.

EXAMPLES

1. Generation of Humanized scFvD9D10

As the use of mouse monoclonals in humans induces a HAMA response, ahumanized antibody or antibody derivative is the alternative. HumanizedscFvD9D10 need to have similar binding and neutralization properties astheir original mouse counterparts, but will elicit hardly any immuneresponse in humans as compared to the parent mouse scFv.

1.1. Modelling

We used computer modelling techniques for the construction of ahumanized scFvD9D10 in order to develop an active scFv with retainedstructure and affinity. The as humanized using a resurfacing strategywhich includes the replacement of ‘non-human’ residues withoutsignificant structural changes of the scFv molecule. This work consistedof 2 main parts. In the first part, a 3D-structure of the mouse scFv wasconstructed. For this purpose, we have homology-modeled D9D10 using IgV_(L) and V_(H) domains with a similar sequence and a known structure.In the second part (the actual humanization step), we have aligned D9D10with similar human sequences to identify ‘typically human residues’.After verifying their structural compatibility with the D9D10 model,they have been proposed as residues-to-be-humanized.

Part 1: 3D-structure of scFvD9D10

Identification of Known Structures with the Most Resembling Sequence

Different BLAST-searches were performed by entering the D9D10 sequenceof either V_(K) or V_(H), by using the ‘BLASTP’ search program and byselecting the Brookhaven Protein Data Bank as the database to besearched. This search was performed 4 times, namely for V_(K) with andwithout CDR-loops and for VH with and without CDR-loops. The obtaineddata are summarized in Table 1.

TABLE 1 Summary of BLAST-search results A) BLAST-search usingD9D10-V_(K) sequence PDB score + CDR score − CDR rank rank Codeident./sim. ident./sim. for V_(H) source I. D. 1 1BAF 87%/92%90%/95% >50 mouse Fab frag. mAb An02 compl. w. its hapten(2,2,6,6-Tetramethyl-1- Piperidinyloxy-Dinitrophenyl) 2 1FOR 80%/90%85%/93% 16 mouse Igg2a Fab frag. (Fab17-Ia) 3 2IFF 78%/86% 84%/90% 15mouse Igg1 Fab Frag. (Hyhel-5) compl. w. Chicken Lysozyme mutant R68K 41FIG 75%/86% 80%/90% 28 mouse Chain L, Immunogl G1 (Kappa Light Chain)Fab′ frag, Mouse 5 1FVB 80%/87% 83%/89% >50 mouse IgA Fv frag.(Anti-Alpha(1->6) Dextran) (Theoret. Model) 6 2HFL 77%/85% 83%/89% 14mouse IgG1 Fab frag. (HyHEL-5) compl. w. Chicken Lysozyme — — — — — — —19  1NCA 60%/73% 70%/84% 1 mouse N9 neuraminidase-NC41 compl. w.Influenza Virus — — — — — — — B) BLAST-search using D9D10-V_(H) sequencePDB score + CDR score − CDR rank rank Code ident./sim. ident./sim. forV_(K) source I. D. 1 1NCA 83%/89% 91%/95% 19   mouse? N9neuraminidase-NC41 compl. w. Influenza Virus 2 1NCB 80%/88% 87%/94% >50  mouse? N9 Neuraminidase-Nc41 Mut. N329D compl. w. Fab, Influenza Virus3 1TET 80%/86% 87%/92% 38 mouse Igg1 Monocl. Fab frag (Te33) compl. w.Cholera Toxin Peptide 3 4 1DBA 80%/87% 86%/92% >50 mouse Fab′ frag. ofthe Db3 Anti- Steroid Monocl. Ab — — — — — — — 16  1FOR 58%/76% 63%/83%2 mouse Igg2a Fab frag. (Fab17-Ia) — — — — — — —

A sequence similarity of more than 70% guarantees a strong structuralsimilarity. For V_(K), at least 6 very good matching structures (allmurine proteins) could be identified: 1BAF, 1FOR, 2IFF, 1FIG, 1FVB and2HFL. The scores for the search with CDR-loops varied from 87% to 77%for identical residues, and from 92% to 85% for chemically similarresidues. The scores for the search without CDR-loops ranged from 90% to83% identical residues and from 95% to 89% similar residues. The smalldifference in homology between the searches with and without CDR-loopssuggests that even some of the CDR-loops are structurally similar. ForV_(H), analogous results were obtained. Four very well matchingstructures could be identified: 1NCA, 1NCB, 1TET and 1DBA with scoresvarying from 83% to 80% identical residues and from 89% to 87% similarresidues when CDR-loops are included. If CDR-loops were not taken intoaccount, significantly higher scores were obtained: from 91% to 86% foridentical residues and 95% to 92% for similar residues. The latter wasdue to the fact that CDR-H3 from D9D10 was not matching well with anysequence.

Three-dimensional Fitting of the Best Candidates

From these scores, it was clear that the V_(K)-fragment from 1FORresembled very well V_(K) from D9D10 (rank nr 2). A reasonably wellhomology was also found for its V_(H) counterpart (rank nr 16). For theheavy domain, 1NCA had a very high score for V_(H) (rank nr 1) and anacceptable score for its V_(K)-domain (rank nr 19). Since the β-barrelsof Fv fragments are well conserved, and since for both V_(K) and V_(H)we dispose of two very good resembling fragments with fairly wellmatching counterparts, we had enough information to start theconstruction of the D9D10 model.

When superimposing (fitting) the complete main chain of 1FOR and 1NCA weobtained a root-mean-square (rms) deviation of 1.1 Å (values around orless than 1 Å indicate a strong structural similarity). Fitting on V_(K)alone gave 1.0 Å and on V_(H) we obtained 0.8 Å. This means that boththe complete structures and the separate V-domains are nearly identical.In order to obtain an even smaller rms-deviation, we fitted all β-standsof the central β-barrel, giving an rms-deviation of 0.52 Å. When theC-terminal strands and certain diverging residues were not taken intoaccount, an rms-deviation as low as 0.37 Å was obtained. The highstructural resemblance of the central β-barrel of both 1FOR and 1NCAensures us that we have correctly positioned the two domains relative toeach other.

In the next step, only the V_(K) fragment of 1FOR and the V_(H) of 1NCAwere retained and CDR-loops of 1FOR and 1NCA were adopted withoutfurther modeling.

Modeling of the D9D10 Sequence onto the Constructed Framework

When the sequences of D9D10 were compared with those of 1FOR-V_(K) and1NCA-V_(H), 21 and 20 mutations were necessary to mutate 1FOR and 1NCAinto D9D10, respectively. These mutations were done simultaneously usingthe Dead-End Elimination method (Desmet et al., 1992) which found theglobally best conformation for all 41 mutations. For both V_(K) andV_(H), the mutations could be done without inducing sterical orenergetical conflicts. As a consequence, we have obtained a veryreliable 3D-model for the variable domains of D9D10 (except for CDR-H3).

PART 2: Humanization of D9D10

Identification of Residues to be Humanized

In order to identify typical D9D10 ‘murine’ residues, V_(K) and V_(H)sequences were again subjected to a BLASTP-search, but this time theentire ‘non-redundant Genbank’ database (PDB+SwissProt+SPupdate+PIR) wassearched for similar sequences. Out of the resulting matches, only humanand humanized sequences were retained and aligned with D9D10.

The alignment revealed several systematic differences in sequencebetween the murine D9D10 molecule and the best matching human V_(K) andV_(H) fragments. From this comparison, we have derived a consensus listof human residues.

Each of these residues was then placed onto the D9D10 model and thefollowing properties were examined: (i) the compatibility with theframework and with neighboring residues, (ii) the solvent accessibilityand (iii) the proximity to the CDR-loops. In general, only D9D10residues which were not found in any human sequence, which werestructurally compatible with the D9D10 framework (and CDR's), and whichwere clearly solvent exposed, were selected for humanization.

For the V_(K) domain we proposed 8 mutations, which were spatiallyclustered into 2 surface patches of 3 residues each plus two isolatedresidues. For the V_(H) domain we pinpointed 9 residues to be humanized.The latter residues formed a surface cluster of 5 residues, one of 2residues and 2 additional isolated residues. For neither of the twodomains, buried residues were retained in the mutation list. The reasonfor this is that we explicitly wanted to preserve the D9D10 frameworkstructure and, also, that buried residues are not ‘visible’ to theimmune system anyway.

Finally, the side-chain conformation of the 8+9 mutations was modeledusing the Dead-End Elimination algorithm. We found that all mutationswere energetically favorable. This strengthened the hypothesis that thehumanization procedure would not affect the antigen binding propertiesof D9D10.

1.2. Construction, Expression, Purification and Evaluation of HumanizedscFvD9D10

Eight substitutions in V_(H)D9D10 and 9 in V_(L)D9D10 had to be carriedout as shown in FIG. 2. Since the different mutations were spread amongthe whole V_(H) and V_(L) sequences, it was decided to assemble thewhole V_(H) and V_(L) sequences out of synthetic oligonucleotides,hereby including all necessary substitutions during the oligonucleotidesynthesis as an alternative to mutagenesis. During the oligonucleotidesynthesis, non-optimal E. coli codons were substituted for more optimalones coding for the same amino acid. Both V_(H) and V_(L) regions wereassembled separately according to the PCR assembly method described byStemmer et al. (1995). The assembled V_(H) and V_(L) regions were firstsubcloned in pGEM-T vectors ( PROMEGA Corp., Madison Wis. US) and theircorrect sequence was confirmed by DNA sequencing. Both humanised regionswere subsequently introduced into the pscFvD9D10H6 expression vector(Froyen et al., 1993). For the assembly of the heavy chain, wesynthesized 18 oligo's, 40 nucleotides in length, which collectivelyencode both strands of the V_(H) region from the AlwNI site to the StyIsite. The plus strand as well as the minus strand consist of 9 oligo'sconfigured in such a way that, upon assembly, complimentary oligo's willoverlap by 20 nucleotides. In these oligo's we included mutations bothleading to “humanised” amino acids at the predetermined sites and to“optimised” E. coli codons.

Oligo No. Oligo Seq. 1s 5′-CGCGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAG-3′(SEQ ID NO 3) 2as 5′-CAGCTGCACCTGGGCCATCGCTGGTTGGGCAGCGAGTAAT-3′ (SEQ IDNO 4) 3s 5′-CGATGGCCCAGGTGCAGCTGGTGCAGAGCGGTAGCGAACT-3′ (SEQ ID NO 5)4as 5′-CGCTCGCACCCGGTTTTTTCAQTTCGCTACCGCTCTGCAC-3′ (SEQ ID NO 6) 5s5′-GAAAAAACCGQGTGCGAGCGTTAAGATCAGCTGCAAAGCG-3′ (SEQ ID N0 7) 6as5′-TCGGTGAAGGTATAACCGCTCGCTTTGCAGCTGATCTTAA-3′ (SEQ ID NO 8) 7s5′-AGCGGTTATACCTTCACCGATTACGGTATGAACTGGGTTA-3′ (SEQ ID NO 9) 8as5′-ACCTTGACCCGGCGCCTGTTTAACCCAGTTCATACCGTAA-3′ (SEQ ID NO 10) 9s5′-AACAGGCGCCGGGTCAAGGTCTGAAATGGATGGGTTGGAT-3′ (SEQ ID NO 11) 10as5′-TTTCACCGGTGTAGGTGTTGATCCAACCCATCCATTTCAG-3′ (SEQ ID NO 12) 11s5′-CAACACCTACACCGGTGAAAGCACCTACGTTGACGATTTC-3′ (SEQ ID NO 13) 12as5′-CTGAAAACGAAACGACCTTTGAAATCGTCAACGTAGGTGC-3′ (SEQ ID NO 14) 13s5′-AAAGGTCGTTTCGTTTTCAGCCTGGATACCAGCGTTAGCG-3′ (SEQ ID NO 15) 14as5′-GCTGATCTGCAGGTAQGCCGCQCTAACGCTGGTATCCAGG-3′ (SEQ ID NO 16) 15s5′-CGGCCTACCTGCAGATCAGCTCTCTGAAAGCGGAAGACAC-3′ (SEQ ID NO 17) 16as5′-GCGCGCAGAAGTAGGTCGCGGTGTCTTCCGCTTTCAGAGA-3′ (SEQ ID NO 18) 17s5′-CGCGACCTACTTCTGCGCGCGTCGCGGTTTCTACGCGATG-3′ (SEQ ID NO 19) 18as5′-GCGCCCTTGGCCCCAGTAATCCATCGCGTAGAAACCGCGAC-3′ (SEQ ID NO 20)

After assembly of the 18 40-mer oligonucleotides, the desired fragmentwas PCR amplified using 2 oligonucleotides complementary to the 5′ and3′ end of the fragment respectively.

Oligo No. Oligo Seq. 1s 5′-CGCGCAGCCGCTGGATTGTTATTAC-3′ (SEQ ID NO 21)2as 5′-GCGCCCTTGGCCCCAGTAATC-3′ (SEQ ID NO 22)

The resulting 381 bp fragment was cloned into a pGEM-T vector, resultingin pGEM-TV_(H)H and several clones were sequenced. A similar approachwas followed for the light chain. Hereby 14 oligos were synthesized, 248-mers and 12 40-mers, which collectively encode both strands of theV_(L) region from the SacI site to the XhoI site. However, since theSacI site was present exactly on an amino acid substitution site, thisrestriction site could not be retained in the synthetic V_(L) gene. Asan alternative, a Bst1107I site was created which will, after ligationwith the blunted SacI site, restore the exact V_(L) reading frame.

Oligo No. Oligo Seq. 1s5′-GCGGTATACTGACCCAGAGCCCGGCGACCATGAGCGCGAGCCCGGGT-3′ (SEQ ID NO 23) 2as5′-CAGGTCAGGGTAACACGTTCACCCGGGCTCGCGCTCATGG-3′ (SEQ ID NO 24) 3s5′-GAACGTGTTACCCTGACCTGCAGCGCGAGCTCTAGCATCA-3′ (SEQ ID NO 25) 4as5′-ATGATACCAGAACATATAGCTGATGCTAGAGCTCGCGCTG-3′ (SEQ ID NO 26) 5s5′-GCTATATGTTCTGGTATCATCAGCGTCCGGGTCAGAGCCC-3′ (SEQ ID NO 27) 6as5′-TATCATAGATCAACAGACGCGGGCTCTGACCCGGACGCTG-3′ (SEQ ID NO 28) 7s5′-GCGTCTGTTGATCTATGATACCAGCAACCTGGCGAGCGGT-3′ (SEQ ID NO 29) 8as5′-CCGCTGAAACGCGCCGGAACACCGCTCGCCAGGTTGCTGG-3′ (SEQ ID NO 30) 9s5′-GTTCCGGCGCGTTTCAGCGGTAGCGGTAGCGGTACCAGCT-3′ (SEQ ID NO 31) 10as5′-ACGGCTGATGGTCAGGCTATAGCTGGTACCGCTACCGCTA-3′ (SEQ ID NO 32) 11s5′-ATAGCCTGACCATCAGCCGTATGGAACCGGAAGATTTCGC-3′ (SEQ ID NO 33) 12as5′-TCTGATGGCAGAAATAGGTCGCGAAATCTTCCGGTTCCAT-3′ (SEQ ID NO 34) 13s5′-GACCTATTTCTGCCATCAGAGCTCTAGCTATCCGTTCACC-3′ (SEQ ID NO 35) 14as5′-CGCGCTCGAGTTTGGTACCCTGACCGAAGGTGAACGGATAGCTAGAGC-3′ (SEQ ID NO 36)

After assembly of the 2 48-mer and 12 40-mer oligonucleotides, thedesired fragment was again PCR amplified using 2 oligonucleotidescomplementary to the 5′ and 3′ end of the fragment respectively.

Oligo No. Oligo Seq. 1s 5′-CGCGGTATACTGACCCAGAGC-3′(SEQ ID NO 37) 2as5′-CGCGCTCGAGTTTGGTACCCTG-3′(SEQ ID NO 38)

The resulting 316 bp fragment was cloned into a pGEM-T vector, resultingin pGEM-TV_(L)H and several clones were sequenced. The assembly PCRprotocol (Stemmer et al., 1995) consisted of 3 steps: gene assembly,gene amplification and cloning. Since single-stranded ends ofcomplementary DNA fragments were filled-in during the gene assemblyprocess, cycling with Taq DNA polymerase resulted in the formation ofincreasingly larger DNA fragments until the full-length gene wasobtained. It can be noted that DNA ligase has not been used in theprocess. After assembly, the desired fragments were amplified using 5′and 3′ end complementary primers. The resulting fragments weresubsequently cloned into a suitable cloning vector such as pGEM-T,giving pGEM-TV_(L)H and pGEM-TV_(H)H. The final vector,pscFvD9D10V_(Hum), was constructed by ligating a 310 bp Bst1107/XhoIfragment originating from vector pGEM-TV_(L)H with a 3180 bpSacIblunt/XhoI fragment originating from vector pscFvD9D10H6V_(H)H(=pscFvD9D10H6 in which V_(H) was replaced by the humanized V_(H)obtained from pGEM-TV_(H)H).

Induction of the humanised scFv D9D10 was carried out in E. coli strainJM83. Detection of His6-tagged scFv's on western blot was done with ananti D9D10 rabbit polyclonal antibody and an anti His6 monoclonalantibody (Babco, Richmond, Calif., USA). Compared to the non-humanizedscFvD9D10 (Froyen et al., 1993), the humanized scFvD9D10 was expressedat approximately 3-5 times higher levels (30-40 mg/l). This increase inexpression level can be due to the fact that during assembly thehumanized scFvD9D10 coding sequence was codon-optimised for E. coliexpression. Alternatively, one or several of the humanized amino acidscan have a beneficial effect on the expression level; or the increase inexpression level can be caused by a combination of the two. As with thenon-humanized scFv, most of the expressed protein was still presentintracellularly (70-80%), with 5-10% present in the periplasmic fractionand 10-20% secreted to the medium.

The cells were harvested and lysed in the presence of proteaseinhibitors at 4° C. by the French press (2 passages at 14.000 psi). Thecell lysate was clarified by centrifugation and the supernatant was usedfor purification. The supernatant was loaded on Zn²⁺-IDA Sepharose FFand the resin was washed by applying an imidazole step gradient. Thedifferent pools were analysed by SDS-PAGE under reducing and nonreducing conditions.

The humanized scFv bound and eluted as expected in the 150 mM imidazoleelution pool and SDS-PAGE showed that the recovered scFv was >90% purein a single step. The shift in relative migration under reducingconditions showed that the scFv was purified in an oxidized form.However, in contrast to the mouse scFv, the humanized scFv showed a hightendency for non specific adsorption, because only 40-50% of the initialproduct was recovered after dialysis.

The humanized scFvD9D10 was shown to have the same biological activityas the mouse scFvD9D10 for neutralizing the antiviral activity of humanIFNγ (described in example 7).

Affinity could be calculated for murine and humanized scFv using SurfacePlasmon Resonance(SPR)-analysis with the BIACORE® (Biacore AB, Uppsala,Sweden). This technology permits real-time mass measurements usingsurface plasmon resonance. SPR is an optical phenomenon, seen as a sharpdip in the intensity of light reflected from a thin metal film coatedonto a glass support. The position of this dip depends on theconcentration of solutes close to the metal surface. In general, aprotein (e.g. antibody) is coupled to the dextran layer (covering thegold film) of a sensor chip and solutions containing differentconcentrations of a binding protein (e.g. antigen) are allowed to flowacross the chip. Binding (association and dissociation) is monitoredwith mass sensitive detection.

In order to determine the affinity of the D9D10 derivatives for hIFNγ,BIACORE® experiments were performed in which the murine D9D10 wasimmobilized onto a CM5 sensorchip (Biacore AB). D9D10 was immobilizedusing amine coupling according to the manufacturer's procedure. Todecrease the non specific interaction of human IFNγ with the carboxylicgroups of the dextran layer, the sensorchip was pretreated with 4 cyclesof EDC/NHS—thus reducing the amount of unblocked carboxylic groupsremaining on the sensor surface—before immobilizing D9D10. Then,immobilization of D9D10 was carried out using a continuous flow of 5l/min on a sensor chip surface initially activated with 17 l of an 0.05MNHS/ 0.2M EDC mixture. 35 L of typically 3 g/ml D9D10 was injected overthe activated surface. Residual unreacted ester groups were blocked byinjecting 17 l of 0.1M ethanolamine pH 8.5. D9D10 was immobiliseddirectly on a CM5 chip at an optimal concentration of 3 g/ml in anacetate buffer pH 5.4 resulting in an immobilization level of about 600RU. Most accurate affinity data were obtained by injecting human IFNγand monitoring the subsequent binding of scFvD9D10; the latterinteracting with remaining free epitopes on human IFNγ. On and off rateswere calculated using the BIAevaluation software (Biacore AB).

Results of a typical experiment are shown in FIG. 3 for murine scFvD9D10and in FIG. 4 for humanized scFvD9D10 (These data were generated inseparate experiments). Calculated data were in good agreement. As offrates were hardly detectable for both constructs in most experiments,only on rates are shown for the concentrations tested. These dataclearly indicated that the humanization did not hamper the bindingcharacteristics of the scFv fragment.

Monoclonal antibodies were generated against the humanized scFvD9D10. Afemale BALB/c mouse was immunized (injected intraperitoneally) 3 timeswith humanized scFvD9D10 (i.e., at days 0 (50 μg), 32(25 μg) and 56(25μg)). Three months after, a final boost of 25 μg was given. Three daysafter this last injection, spleen cells were retrieved from theimmunized mouse and used for cell fusion. Dissociated splenocytes fromthe immunized mouse were fused with murine myeloma cells SP2/0-Ag14(ATCC, CRL-1581) at a ratio of 10:3 using a polyethylene glycol/DMSOsolution mainly according the procedure as described by Köhler andMilstein (1975). The fused cells were mixed up and resuspended in DMEMmedium supplemented with hypoxanthine, sodium pyruvate, glutamine, anon-essential amino acid solution, 20% heat-inactivated fetalclone(Hyclone Lab., Utah) and 10% BM-Condimed (Boehringer Mannheim). Thecells were then distributed to 96 well plates to which aminopterin wasadded 24 hours after the cell fusion. Each well contained between 1 to 5growing hybridoma clones at the average. After 8 days supernatant of thewells was collected and screened in an ELISA for binding to humanizedscFvD9D10. The antibodies of the hybridomas thus generated were furthertested for their binding capacity to murine and humanized scFvD9D10 andhuman IgG. Certain monoclonal antibodies derived from this hyper immunemouse did recognize not only humanized scFvD9D10 but also human IgG,indicating the quality of the humanization strategy. Using theantibodies which specifically interact with humanized scFvD9D10 (1D5C5;11E2G6; 10F12A2 available at Innogenetics N.V., Industriepark Zwijnaarde7, Box 4, B-9052 Ghent, Belgium) and do not cross react with the yettested human IgG preparations, an ELISA is generated for detecting andquantifying D9D10 derived constructs in human and primate serum.

Immunization experiments in rabbit and mouse with his-tagged proteinsincluding the humanized scFvD9D10 revealed weak to fairly highimmunogenic responses of the his tail. Consequently, we made a newconstruct and removed the C-terminal hexahistidinetag from the scFvD9D10(humanized scFvD9D10 H6⁻). This was done by cutting vectorpscFvD9D10V_(Hum) with XhoI and EcoRI and substituting the His6-tailwith a tandem stop codon and a unique NcoI site for easy identification.This was accomplished using two synthetic oligo's (oligo1:5′-TCGAGATCAAACGGTAATAGCCATGG-3′ (SEQ ID NO 39); oligo2:5′-AATTCCATGGCTATTACCGTTTGATC-3′ (SEQ ID NO 40)) which, when annealed,reconstitute the D9D10 V_(L) coding sequence, followed by tandem stopcodons and a unique NcoI site for identification. The annealeddouble-stranded oligo has sticky ends corresponding to a XhoI site atthe 5′ end and EcoRI site at the 3′ end. The oligo was ligated into theXhoI/EcoRI opened pscFvD9D10V_(Hum) vector resulting inpscFvD9D10V_(Hum)[H6⁻]. Expression analysis showed identical expressionlevels and localisation compared to the His6-tagged D9D10 in E. coli.

2. Generation of Humanized, Chimeric D9D10

Two fusion cDNA-genes respectively coding for the heavy and light chainfusion-proteins of the humanized D9D10 whole antibody were constructed.The light chain fusion cDNA consists of the cDNA encoding the mouseD9D10 light chain leader sequence (Ldr), needed for efficient transportof the fusion protein in the host cell, the humanized D9D10 light chainvariable domain cDNA (V_(Lh)), followed by a human immunoglobulinkappa-light chain constant domain (C_(L)).

The heavy chain fusion cDNA consists of the mouse D9D10 light chainleader cDNA-sequence (Ldr), followed by the humanized D9D10 heavy chainvariable domain cDNA (V_(Hh)) and a human IgG1 heavy chain constantdomain (C_(H)=C_(H)1-Hinge-C_(H)2-C_(H)3) cDNA, in which theC1q-complement binding site in the C_(H)2 region, known to inducecomplement activation upon injection of the recombinant antibody, wasmutated (Pro_(33l)→Ser) (Xu et al., 1994).

PCR Cloning of Human Immunoglobulin Cγ1 and C_(K) cDNA

Total RNA was isolated from human tonsil cells (frozen pellet of ±10⁷cells) following the Chomczynski GuSCN/acid phenol isolation method(Chomczynski and Sacchi, 1987). 140 μg total RNA was obtained. cDNA wasprepared by annealing 700 ng total RNA to 300 ng random hexamers(Pharmacia, Upsala, Sweden) and reverse transcription for 90 min at 42°C. using AMV reverse transcriptase (RT-Stratagene) in a final volume of20 μl (50 mM Tris pH 8.3, 40 mM KCl, 6 mM MgCl2, 5 mM DTT). The reactionwas inactivated by heating at 90° C. for 15 min.

Cloning of the Human CK cDNA:

The cDNA was used as template for PCR amplification of the human C_(K)cDNA using primer sequences based on the Genbank database sequence ,accession # V00557 and # J00241.

oligo #7061 (C sense primer): (SEQ ID NO 41)               ThrValAla...5′-TCGAAGCTT AGTACTGTGGCTGCACCATCTGT-3′        HindIII ScaI oligo #7060(C_(K) antisense primer): (SEQ ID NO 42)                CysGluGly...5′-GTCGAATTC TfGCGCACTCTCCCCTGTTGAAGC-3′         EcoRI FspI

PCR amplification using the 7060/7061 primers is expected to yield afragment of 342 basepairs. ScaI/FspI digestion of this fragment shouldyield a blunt fragment starting at the first AA, Thr of C_(K) and endingat the last AA, Cys. A stop codon is not present.

PCR reaction was carried out in a final volume of 50 μl, using 2 μl ofthe RT reaction, 10 pmol of each primer and 5U of either Taq DNApolymerase (Stratagene, La Jolla, Calif., USA). dNTPs were present at afinal concentration of 200 μM in 1×Taq buffer as provided by thesupplier. Reactions were overlaid with 75 μl paraffin oil. Cyclingconditions were as follows. After an initial denaturation of 5 min at95° C. 40 PCR cycles (1 min 94° C., 1 min at appropriate annealingtemperature of 60° C. and 1 min at 72° C.) were carried out. There was afinal extension phase of 10 min at 72° C. 5 μl amounts of the reactionwere run on agarose gels.

The PCR reaction with the 7060/7061 primer pair yielded a single band of±300 bases, which was purified using the Geneclean® kit (Bio101, Vista,Calif, USA), digested with EcoRI/HindIII, phenol:CHCl₃ extracted andligated into EcoRI/HindIII digested pBSK(−) vector (Stratagene). Theligation mix was electroporated into the DH5αF′ bacterial strain.Transformed bacteria were plated onto X-gal/IPTG LB agar plates forblue/ white selection of recombinants. Four white colonies were selectedfor further analysis and plasmid DNA was prepared. EcoRI/HindIIIrestriction analysis showed that all 4 C_(K) transformants contained aninsert of the correct length. The 4 inserts were entirely sequenced. Oneclone was completely identical to the database sequence (accession nrsV00557 and J00241). The corresponding plasmid was named pBLSKIGkappaC.

Cloning of the Human Cγ1 Heavy Chain Constant Domain cDNA:

The cDNA was used as template for PCR amplification using primersequences based on the Genbank database sequence: accession # Z17370.

oligo #7601 (Cγ1 sense primer; 48-mer, should only be Cγ1 specific)               AlaSerThr... 5′-CTAGAATTCTGCGCATCCACCAAGGGCCCATCGGTCTTCCCCCTGGCA-3′ (SEQ ID NO 43)         Ec6RIFspI oligo #7600 (C 1 antisense primer):                LysGlyProSer...5′-GTAAAGCTT GAGCTCTTACCCGGAGACAGGGAGAGG-3′ (SEQ ID NO 44)       HindIII SacI

PCR amplification using the 7601/7600 primer couple is expected to yielda fragment of 1016 basepairs. FspI/SacI cleavage of this fragmentfollowed by removal of the SacI 3′ overhang should yield a bluntfragment starting with the first AA, Ala of Cγ1 and ending with the lastAA, Lys. A stop codon is not included. PCR reactions were carried out ina final volume of 50 μl, using 2 μl of the RT reaction, 10 pmol of eachprimer and 5U of Taq DNA polymerase (Stratagene). dNTPs were present ata final concentration of 200 μM in 1×Taq buffer as provided by thesupplier. Reactions were overlaid with 75 μl paraffin oil. Cyclingconditions were as follows: after an initial denaturation of 5 min at95° C. 40 PCR cycles (1 min 94° C., 1 min at appropriate annealing temp.55° C. and 1 min at 72° C.) were carried out. There was a finalextension phase of 10 min at 72° C. 10 μl amounts of the reaction wererun on agarose gels. A single band of around 1 kb was obtained. The 1 kbband, obtained with the 7601/7600 primer pair, was purified using theQiaquick™-kit (Qiagen, Hilden, Germany) and ligated into pGEM-T-vector.The ligation mix was transformed into the DH5αF′ bacterial strain.Transformed bacteria were plated onto X-gal/IPTG LB agar plates forblue/white selection of recombinants.

Eight white colonies were selected for further analysis and plasmid DNAwas prepared. Restriction analysis with BstXI (=specific for IgG-1;absent in IgG-2) showed that 6 transformants contained an Cγ1 insert ofthe correct length. One clone was entirely sequenced and was shown to beidentical to the database sequence, except for 3 codon switches, wichcorrespond to a described allotypic variant Gm(−1,4) of the human IgG1(lys214−>arg214, asp356−>glu356 and leu358−>met358 respectively). Sincethe Gm(−1) (“nonmarker”), glu356/met358, also occurs on Cγ2, this markerwill likely not be immunogenic when introduced in humans. The clonedsequence also contained two silent base switches in comparison to thedatabase sequence Z17370. The final construct was named pGEMThIGG1c.

The C1q-complement binding site present in the C_(H)2 region of thehuman IgG1, known to induce complement activation upon injection of therecombinant antibody (Xu et al., 1994), was later mutated (Pro₃₃₁→Ser)as described further during the assembly of the humanized D9D10 fusioncDNA.

Construction of Fusion cDNAs

In order to assemble the light—and heavy chain fusion genes, severalintermediate cloning constructs, generated by PCR-assembly andamplification, were needed.

Assembly of the Light Chain Fusion cDNA

The mouse D9D10 V_(K) leader sequence cDNA was cloned by PCR-assembly(Stemmer et al., 1995) of four partially overlapping syntheticoligonucleotides [IG8180, IG8179, IG8178 and IG8176] of each 40 bps, andsubsequent PCR-amplification with two specific outside primers [IG 8175and 8174]. The resulting 100 bp PCR fragment I, named Ldr, consist of a5′ untranslated region of 20 bp , including an XbaI cloning site, andthe cDNA encoding the complete D9D10 V_(K) leader peptide (20 AA) and 20bp of the humanized D9D10 light chain variable domain cDNA encoding thefirst 6 AA.

Sense strand oligos:                   XbaI IG81805′-GTCCCCCGGGTACCTCTAGAATGGATTTTCAAGTGCAGAT-3′ (SEQ ID NO 45) IG81795′-TTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATACTCTCG-3′ (SEQ ID NO 46) Antisensestrand oligos: IG8178 5′-CTCTGGGTCAGCTCGATGTC6GAGAGTATGACTGAGGCAC-3′(SEQ ID NO 47) IG8176 5′-TGATTAGCAGGAAGCTGAAAATCTGCACTTGAAAATCCAT-3′(SEQ ID NO 48) PCR amplification primers:                   XbaI IG8175(sense) 5′-GTCCCCCGGGTACCTCTAGAATG-3′ (SEQ ID NO 49) IG8174 (antisense)5′-CTCTGGGTCAGCTCGATGTCC-3′ (SEQ ID NO 50) IG81756      IG8180                    IG8179------------------- ------------------                 IG8176         IG8178           ----------------- -----------------                                       IG81747

The humanised light chain variable domain as present in pGEM-T-V_(L) H,described earlier, was PCR-amplified using primers [IG8172 and IG8171]designed to produce PCR fragment II containing the complete variabledomain cDNA with exception of the last 3 amino acids (IKR), and flankedat the 3′-terminus by an XhoI-cloning site.

IG8172 (sense) 5′-GACATCGAGCTGACCCAGAGCCCGGCG-3′ (SEQ ID NO 51)        XhoI IG8171 (anti- 5′-CGCGCTCGAGTTTGGTACCCTG-3′ sense) (SEQ IDNO 52)

Fusion of the two DNA fragments PCR-I (Ldr) and PCR-II (V_(Lh)), having20 bp overlap, was performed by overlap PCR using primerset IG8175 andIG8171. The resulting PCR-III fragment was directly cloned in pGEM-Tresulting in the pGEMLdrV_(Lh) plasmid.

                  XbaI IG8175 (sense) 5′-GTCCCCCGGGTACCTCTAGAATG-3′ (SEQID NO 49)         XhoI IG8171 (antisense) 5′-CGCGCTCGAGTTTGGTACCCTG-3′(SEQ ID NO 52)

The human _(K)-light chain constant domain was cloned byPCR-amplification using pBLSKIGkappaC as template with primers IG8170and IG8169. The resulting PCR-IV fragment consists of the cDNA sequenceencoding the last 3 AA of V_(Lh) and the complete human Ckappa constantdomain, followed by a stop codon and an EcoRI cloning site. The PCR-IVDNA was directly cloned in the pGEM-T vector resulting in thepGEM-TC_(L) plasmid.

        XhoI IG8170(sense) 5′-GCGCCTCGAGATCAAACGGACTGTGGCTGCACCATCTG-3′(SEQ ID NO 53)        EcoRI IG8169(antisense)5′-GCCGGAATTCCTAGCACTCTCCCCTGTTGAAG-3′ (SEQ ID NO 54)

Fusion of LdrV_(Lh) and C_(L) cDNA in the pGEM-T backbone was realisedby insertion of the C_(L)-containing XhoI-SpeI fragment, isolated frompGEM-TC_(L) plasmid, in the pGEMLdrV_(Lh) plasmid. The resultingconstruct was named pGEMhD9D10_(L).

Assembly of the Heavy Chain Fusion cDNA

The mouse D9D10 V_(K) leader sequence cDNA was cloned by PCR-assembly(Stemmer et al., 1995) of four partially overlapping syntheticoligonucleotides [IG8180, IG8179, IG8176 and IG8177] of each 40 bps, andsubsequent PCR-amplification with two specific outside primers [IG 8175and 8173]. The resulting 100 bp PCR-V fragment, named Ldr-2, consist ofa 5′ untranslated region of 20 bp, including an XbaI cloning site, andthe cDNA encoding the complete D9D10 V_(K) leader peptide (20 AA) and 20bp of the humanized D9D10 heavy chain variable domain cDNA encoding thefirst 6 AA.

Sense strand oligos:                    XbaI IG81805′-GTCCCCCGGGTACCTCTAGAATGGATTTTCAAGTGCAGAT-3′ (SEQ ID NO 45) IG81795′-TTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATACTCTCG-3′ (SEQ ID NO 46) Antisensestrand oligos IG8177 5′-CTCTGCACCAGCTGCACCTGCGAGAGTATGACTGAGGCAC-3′ (SEQID NO 55) IG8176 5′-TGATTAGCAGGAAGCTGAAAATCTGCACTTGAAAATCCAT-3′ (SEQ IDNO 48) PCR amplification primers:                   XbaI IG8175(sense)5′-GTCCCCCGGGTACCTCTAGAATG-3′ (SEQ ID NO 49) IG8173(antisense)5′-CTCTGCACCAGCTGCACCTGC-3′ (SEQ ID NQ 56) IG81756      IG8180                 IG8179------------------- ------------------                 IG8176         IG8177           ----------------- -----------------                                       IG81737

The humanised variable heavy chain domain as present in pGEM-T-V_(H)H,described earlier, was PCR-amplified using primers (IG8168 and IG8167)designed to produce PCR-VI fragment containing the complete variabledomain cDNA, and flanked at the 3′-terminus by an XhoI-cloning site.

IG8168(sense)

IG8168(sense) 5′-CAGGTGCAGCTGGTGCAGAGCGGTAG-3′ (SEQ ID NO 57)          XhoI IG8167(antisense)5′-CGCCGGCTCGAGACGGTGACCGTGGTCCCTTGGCCCCAGTAATCC-3′ (SEQ ID NO 58)

Fusion of Ldr-2 and V_(Hh) was performed by overlap PCR on a mixture ofPCR-V and PCR-VI using sense primer IG 8175 and an antisense primer IG8166, resulted in a PCR fragment (LdrV_(Hh)) which was directly clonedin a pGEM-T vector , resulting in PGEMLdrV_(Hh).

                  XbaI IG8175(sense) 5′-GTCCCCCGGGTACCTCTAGAATG-3′ (SEQID NO 49)           XhoI IG8166(antisense) 5′-CGCCGGCTCGAGACGGTGACC-3′(SEQ ID NO 59)

The human heavy chain constant domain cDNA was produced by PCRamplification on pGEMThIGG1c as template, using sense primer IG 8165,designed to introduce a XhoI restriction site and antisense primer IG8164 that added an extra leucine to the C_(H) sequence and introduced aSTOP codon followed by an EcoRI cloning site. The introduction of acodon for a leucine provided, together with the codon for a lysine(normally the last amino acid), a HindIII restriction site. This HindIIIsite was used to insert a scFv-module (cfr MoTAbII expression plasmids,see below). The resulting fragment PCR-VII was inserted in the pGEM-Tvector resulting in plasmid pGEM-TC_(H).

               XhoI IG8165(sense) 5′-GCCGCTCGAGCGCATCCACCAAGGGC-3′ (SEQID NO 60)               EcoRI    HindIII IG8164(antisense)5′-GCCGGAATTCGCTAAAGCTTACCCGGAGACAGGGAGAGG-3′ (SEQ ID NO 61)

The amino acid Pro at position 331 in the C_(H)2 domain of both IgG1 andIgG4 immunoglobulins is described to contribute to their differentialability to bind and activate complement (Xu et al., 1994). ThePro331-codon CCC was therefore mutated to a Ser331-codon, TCC. Twospecific primers IG 8460 and IG8459 were designed, to introduce thismutation by PCR mutagenesis.

Two separate PCR-amplifications were performed on pGEM-T-C_(H) astemplate using (1) primers IG2617, matching with the T7-promoter regionin pGEM-T and IG8460, resulting in a 733 bp PCR-VIII fragment, and (2)primers IG 8459 and IG3899, matching the SP6-promoter in pGEM-T,resulting in a 473 bp PCR-IX fragment. Overlap PCR was subsequentlyperformed on a mixture of PCR-VIII and PCR-IX, using again the primersIG2617 and IG3899, resulting in a 1178 bp PCR-X fragment. The amplifiedPCR-X fragment was eventually inserted as an XhoI-SpeI fragment (1018bp) in the pGEMLdrV_(Hh) plasmid. The resulting pGEMhD9D10_(H) plasmidcontains the complete coding sequence of the humanized D9D10 heavy chainfusion protein.

IG8459 (sense) 5′-GCCCTCCCAGCCTCCATCGAGAAAAC-3′                Ser₃₃₁(SEQ ID NO 62) IG8460 (antisense) 5′-GTTTTCTCGATGGAGGCTGGGAGGGC-3′                Ser₃₃₁ (SEQ ID NO 63) IG2617 (sense-T7)5′-TAATACGACTCACTA-3′ (SEQ ID NO 64) IG3899 (antisense-SP6)5′-ATTTAGGTGACACTATAG-3′ (SEQ ID NO 65) *Construction of mammalianexpression plasmids

Successful high level expression of recombinant immunoglobulins has beenreported in both lymphoid and non-lymphoid mammalian cell lines.Basically an expression plasmid(s), containing the immunoglobulin genescoding for respectively heavy and light chain proteins undertranscriptional control of a promoter/enhancer unit recognized inmammalian cells, is introduced in the chosen host cells together with(as one plasmid or on separate plasmids) a drug-resistance geneexpression unit by classical cell transfection techniques. Cells thathave randomly integrated the foreign expression units in their cellgenome are initially selected for their drug-resistant phenotype andsecondly for high level, stable expression of the protein of interest,the immunoglobulin. After gene integration, an increase in theimmunoglobulin expression level can be obtained by coamplification ofthe genes through further selection of isolated recombinant cell linesfor increased resistance to the drug resistance marker.

One possible example of a successful strategy for mammalian cellexpression is the glutamine synthetase based selection/amplificationmethod shown to result in high level production of mammalian proteins indifferent cell types including Chinese hamster ovary cells (CHO)(Cockett et al., 1990) and myeloma cells, Ns0 (Bebbington et al., 1992).The use of the system is covered by patents WO87/04462 and WO89/10404(Lonza Biologicals, Slough, UK).

Following the GS-expression method, the fusion genes coding forrespectively the heavy- and light chain of the recombinantimmunoglobulins were cloned in a mammalian expression plasmid (pEE12 orpEE14) under transcriptional control of the strong Cytomegalovirus majorimmediate early promoter /enhancer (CMV-MIE). This plasmid also carriesa cloned glutamine synthetase (GS) gene expression element that can actas a dominant selectable marker in a variety of cells. GS indeedprovides the only pathway for synthesis of glutamine using glutamate andammonia as substrates. The final fusion product LdrV_(Lh)C_(L) orhD9D10_(L) was directly cloned as an XbaI-EcoRI fragment isolated fromthe plasmid pGEMhD9D10_(L) in the mammalian expression vectors pEE14(for CHO) and pEE12 (for Ns0) (Lonza biologicals) under transcriptionalcontrol of the CMV promoter, resulting in the plasmids pEE12hD9D10_(L)and pEE14hD9D10_(L).

The cDNA encoding the heavy chain fusion protein LdrV_(Hh)C_(H) orhD9D10_(H) was first transferred from the pGEMhD9D10_(H) construct as anXbaI-EcoRI fragment in the intermediate vector pEE6hCMV-BgIII (LonzaBiologicals), also behind the CMV promoter. From the latter constructpEE6hD9D10_(H) a complete mammalian expression casette, consisting ofCMV-promoter followed by the fusion gene and a polyadenylation site,were transferred as an BgIII-BamHI DNA fragment in the BamHI openedplasmids pEE12hD9D10_(L) and pEE14hD9D10_(L) expression plasmids alreadyavailable. The final expression plasmids, named pEE12hD9D10 andpEE14hD9D10 then consists of the pEE-backbone plasmid containing theGS-selection unit, carrying the light chain fusion gene expressioncasette followed by a comparable heavy chain fusion gene expressioncasette.

The approach of assembling a single expression plasmid containingseparate transcription units for both heavy and light chains and theselectable marker, is adviced in order to ensure coamplification withthe marker gene.

A schematic representation of both plasmids is given in FIGS. 5 and 6.

The cDNA sequence encoding the complete humanized D9D10 heavy chainfusion protein is given in FIG. 7. (SEQ ID NO 66)

The cDNA sequence encoding the humanized D9D10 light chain fusionprotein is given in FIG. 8. (SEQ ID NO 68)

The amino acid sequence of the humanized D9D10 heavy chain fusionprotein is given in FIG. 9. (SEQ ID NO 67)

The aminoacid sequence of the humanized D9D10 light chain fusion proteinis given in FIG. 10. (SEQ ID NO 69)

Small Scale Expression of Humanized D9D10 Chimeric Antibody in COS Cells

A quick way to determine the feasibility of expressing a recombinantprotein in mammalian cells and to evaluate its functionality, istransient expression of the product in COS cells (Gluzmann, 1981). COScells are Simian Virus 40 (SV40)-permissive CV1 cells (African monkeykidney) stably transformed with an origin-defective SV40 genome, therebyconstitutively producing the SV40 T-antigen. In SV40-permissive cells,T-antigen initiates high copy number transient episomal replication ofany DNA-vector that contains the SV40 origin of DNA replication. Boththe pEE12 and pEE14 expression vectors contain an SV40 origin ofreplication in the SV40 early promoter region controlling theGS-selection gene, and thus permits efficient transient expression inCOS cells.

Small amounts of functionally active antibody were made by transientexpression in COS cells. COS7 cells (ATCC CRL 1651) were routinelycultured in DMEM supplemented with 0.03% glutamine and 10% fetal calfserum. For preparative scale transfection, an optimizedDEAE-transfection protocol (McCutchan, 1968) was used. Alternatively,other well known transfection methods such as Ca-phosphateprecipitation, electroporation, liposome-based transfection can be used.Briefly, exponentially growing COS7 cells were seeded in cell factories(Nunc, Rochester, N.Y., USA) at 3.5 10⁴ cells/cm² about 18 h beforetransfection, after which the cells were washed twice with MEM-Hepes pH7.1 (Gibco, Rockville, Md., USA) and allowed to cool to benchtemperature. 0.5 μg/cm² cell surface of high quality plasmid DNA(CsCl-density purification) of the mammalian expression plasmidspEE12hD9D10 and pEE14hD9D10 was ethanol precipitated, redissolved in 25μl/cm² MEM-Hepes pH 7.1 and slowly added to the same volume of 2 mg/mlDEAE-dextran MW 500.000 (Pharmacia) in MEM-Hepes pH 7.1. TheDNA-DEAE-dextran precipitate (50 μl/cm²) was allowed to form for 20-25min, put on the cells for 25 min and removed to be stored at −20° C.(the same precipitate can be reused in a second transfection experimentwith the same efficiency).

The cells were incubated during the next 3.5 hours in DMEM growth medium(Gibco) containing 0.1 mM chloroquine (Sigma) (0,3 ml /cm²) in aCO₂-incubator at 37° C., then washed two times with growth medium andfurther incubated for 18 hrs in complete culture medium enriched with0.1 mM sodium butyrate (Sigma) at 37° C. (0.3 ml/cm²). The next day thecells were washed twice with serum free DMEM medium supplemented with0.03% glutamine (Merck) and then incubated for 48h (determined inanalytical scale experiments as the optimal harvest time) in 150 μl/cm²cell surface of the same medium at 37° C., after which conditionedmedium was harvested and stored at −70° C. until purification. Asnegative control COS cells were also transfected with the emptyexpression vectors pEE12 and pEE14.

Quality control of the crude CM was performed by IFNγ-binding assay inELISA format, by SPR-analysis and by measuring the inhibition of IFNγmediated MHC class II-induction.

Human Interferon—Coating Elisa

96 well ELISA culture plates (Nunc 469914) were coated with 100 ng/wellhIFNγ (Genzyme 80-3348-01, 1 mg/ml) diluted in 50 mM TrisHCl pH8.5, 150mM NaCl, by 18 h incubation at 4° C. Blocking of nonspecific binding wasperformed in PBS/0.1% caseine (×200 μl/well, 1 h, 37° C.). All washingsteps were performed with PBS/0.05% Tween-20 (3×200 μl/well). Purifiedmouse-human chimeric D9D10 whole antibody (EP 0 528 469 to Billiau andFroyen), produced by transient expression in COS cells, was used aspositive control (concentration range 500 ng/well to 4 ng/well, ½dilution series prepared in the sample diluent, 100 μl/well). Sampleswere diluted in a ½ dilution series in PBS/0.1% caseine, and incubatedfor 2 h at 37° C. Detection was performed using an alkaline-phophataseconjugated goat-anti-human IgG_(H+L) (PromegaS3821), diluted 1/2000 inPBS/0.05% caseine, incubated for 2 h at 37° C. AP-substrate(SigmaN-2765) was used at a concentration of 1 mg/ml in 100 mM TrisClpH9.5, 100 MM NaCl, 5 mM MgCl₂. Plates were analysed at 405/595 nm afterresp. 15 and 30 min incubation at 37° C.

Results are shown in FIG. 11: humanized D9D10 clearly interacts withhuman IFNγ coated onto the wells.

SPR Analysis

A comparable set up was used as described for the evaluation of themurine and humanized scFvD9D10 derivatives. Briefly, murine D9D10 wasimmobilized directly onto a B1 sensorchip (BIACORE AB)—containing lesscarboxylic groups and for which as such no pretreatment is necessary—ata concentration of 10 μg/ml D9D10 in an acetate buffer pH 4.8 usingamine coupling. A fixed concentration of 8 μg/ml human IFNγ was added,followed by the injection of either murine D9D10 (10 μg/ml; positivecontrol) or crude COS supernatant containing humanized D9D10. Resultsare shown in FIG. 12. These data clearly illustrate the presence ofactive, IFNγ binding molecules in the COS supernatant. As no exactconcentrations were determined of the humanized D9D10, no affinity datawere calculated.

Inhibition of MHC Class II-induction

See Example 8.1.

Purification of Humanized D9D10

Humanized D9D10 was purified using classical protein A chromatography(Perry and Kirby, 1990; Page and Thorpe, 1996). Quality control of thepurified antibody construct was performed by Western Blot (classicaltechnology) and ELISA. The latter is done as described above and resultsare shown in FIG. 13. From these results it is clear that purified,humanized D9D10 is specifically interacting with IFNγ coated onto thewells.

Generation of Stable Mammalian Expression Cell Lines

For generation of stable mammalian expression cell line, two host celllines Ns0 (Galfre and Milstein, 1981; ECACC 85110503) and CHO-K1 (ATCCCCL61) were used.

The glutamine-dependent NS0 cells were routinely cultured in Lonza DME(JRH 51435)/200 mMglutamine/10%FCS. High quality plasmid DNApEE12hD9D10, prepared by CsCl-density purification, and linearized bySalI digestion, was used for transfection of the NS0 cells byelectroporation (40 μg DNA/10⁷ cells). Transfected cells were thenselected for the glutamine-independent phenotype by gradual reducing theglutamine concentration. Selection was performed in Lonza DME(JRH51435)/GS supplement (JRH58672)/10% dialysed FCS. Individual NS0clones were isolated after±2 weeks of selection. The clones wereanalysed for recombinant antibody production and secretion by testingthe cell conditioned medium in the IFNγ-coating ELISA described earlier.

Several positive cell lines were selected for subsequent vectoramplification by growth in the presence of the GS-inhibitor MSX(methionine sulfoximine), resulting in increased humanized D9D10antibody expression levels.

Large scale production of the recombinant antibody using high expressingNS0 recombinant cell lines is done in bioreactor systems (e.g. hollowfibre systems).

CHO-K1 cells were routinely cultured in GMEM-S (JRH51492)/200 mMglutamine/10%FCS. High quality plasmid DNA pEE14hD9D10, prepared byCsCl-density purification, was directly used for transfection of CHO-K1cells by Ca²⁺-phosphate transfection techniques (12μg/1.15 10⁶ cellsseeded 18 h before transfection on T-flasks). Selective medium,GMEM-S(JR51492)/GS supplement (JRH58672)/10% dialysed FCS/25 M MSX wasadded to the cells 24 h post-transfection. Individual clones could beisolated±2 weeks after transfection. Selected clones were analysed forrecombinant antibody expression and secretion by testing the cellconditioned medium in the IFNγ-coating Elisa described earlier. Severalpositive cell lines were selected for subsequent vector amplification bygrowth in the presence of increased concentrations of the GS-inhibitorMSX, resulting in increased antibody expression levels.

Large scale production of the recombinant antibody using high expressingCHO-K1 recombinant cell lines is done in bioreactor systems (e.g. hollowfibre or ceramic core systems).

3. Generation of Humanized Sheep Anti-IFNγ Antibodies

Sheep antibodies were generated by immunizing sheeps according tostandard immunization protocols. Briefly, sheeps were injectedintradermally on multiple sites with the antigen (recombinant humanIFNγ(procaryotic origin)) for several times over a timeframe of severalmonths (day 0, 14, 28, 56, extra injections on a monthly basis). Serumis tested for its antiviral activity and its affinity (using SPRanalysis).

As elution conditions necessary to elute an antigen from its antibodyreflect the affinity of the antibody (McCloskey et al., 1997),experiments are performed in which the elution conditions of the sheepantibodies for human IFNγ were compared with those of the scFvD9D10antibody.

Sheep monoclonal antibodies are generated by fusing B-lymphocytesisolated from peripheral blood with murine Sp2/0 myeloma cells accordingto the protocol as described in example 1. The affinity of theantibodies for human IFNγ is determined by SPR analysis as described inexample 1.

4. Generation of Aanti-IFNγ Tetravalent Antibody Constructs

4.1. Generation of MoTAb I

The MoTAb I (Monospecific Tetravalent Antibody) molecule is defined as amolecule which consists of 4 identical scFv molecules (e.g. humanizedD9D10 scFv's) in the format of a homodimer of two identical molecules,each containing two scFv's. Both scFv's are linked together using adimerisation domain, which drives the homodimerisation of the molecule(see FIG. 1). Comparable structures have already been described (Pack etal., 1995, Plückthun & Pack, 1997).

The humanized D9D10 scFv was used as a building block to generate theMoTAbI molecule using standard recombinant DNA techniques. A singleMoTAb subunit started with a humanized D9D10 scFv followed by adimerisation domain flanked by flexible linkers. The dimerisation domainwas in turn linked C-terminally to a second D9D10 scFv. Finally adetection and purification tag was added to the extreme C-terminus ofthe molecule. However, in order to circumvent possible immunologicalreactions against the tag, MoTAb I was also produced in an untaggedversion. The sequence coding for the dimerisation domain and theflanking linkers were made synthetically using the method described byStemmer et al. (1995). This synthetic domain was subsequently linked toboth D9D10 scFv's. As linkers between the dimerisation domain and thescFv's, we have used the flexible and proteolysis-resistant truncatedhuman IgG3 upper hinge region (Pack & Plückthun, 1992). As dimerisationdomain we used either the helix-turn-helix motif described by Pack etal. (1993) or the leucine-zipper dimerisation domain originating fromthe human JEM-1 protein as described by Duprez et al. (1997).Optionally, an additional cysteine residue is inserted next to thedimerisation domain to provide extra stability. When applicable, aC-terminal detection and purification tag e.g. a hexahistidine sequence,is used. The sequences were assembled in such a way that functionaldomains were easily replaceable using unique restriction sites presentin the molecule. For the construction of the pGEM-THDH vector, wesynthesized 10 oligo's which collectively encode both strands of the HDHregion (hinge region-dimerization domain-hinge region) flanked by a XhoIand a SpeI restriction site. The plus strand as well as the minus strandconsist of 5 oligo's configured in such a way that, upon assembly,complimentary oligo's will overlap by 20 nucleotides. In these oligo'sthe codons where optimised for optimal E.coli usage. The resulting 223bp fragment was cloned into a pGEM-T vector and several clones weresequenced.

Assembly Oligonucleotides for the HDH-domain

Oligo No. Oligo Seq. 1s 5′-CGCGCTCGAGATCAAACGGACCCCGCTGGGTGATACCACTC-3′(SEQ ID NO 70) 2as 5′-CAGTTCACCTCCGGAGGTATGAGTGGTATCACCCAGCGGG-3′ (SEQID NO 71) 3s 5′-ATACCTCCGGAGGTGAACTGGAAGAGCTGTTGAAACATCT-3′ (SEQ ID NO72) 4as 5′-GACCTTTCAGCAGTTCTTTCAGATGTTTCAACAGCTCTTC-3′ (SEQ ID NO 73) 5s5′-GAAAGAACTGCTGAAAGGTCCGCGGAAAGGTGAACTGGAG-3′ (SEQ ID NO 74) 6as5′-TTCAGGTGCTTCAGCAATTCCTCCAGTTCACCTTTCCGCG-3′ (SEQ ID NO 75) 7s5′-GAATTGCTGAAGCACCTGAAAGAGCTGTTGAAAGGTACCC-3′ (SEQ ID NO 76) 8as5′-ATGGGTAGTATCACCTAGGGGGGTACCTTTCAACAGCTCT-3′ (SEQ ID NO 77) 9s5′-CCCTAGGTGATACTACCCATACCAGCGGTCAGGTGCAACT-3′ (SEQ ID NO 78) 10as5′-CGCGGAATTCGCGTTCGCGACTAGTTGCACCTGACCGCTGGT-3′ (SEQ ID NO 79)

Amplification Oligonucleotides for the HDH-domain:

Oligo No. Oligo Seq. 1s 5′-CGCGGTATACTGACCCAGAGC-3′ (SEQ ID NO 80) 2as5′-CGCGCTCGAGTTTGGTACCCTG-3′ (SEQ ID NO 81)

The MoTAbI expressionplasmid was constructed as followed: The scFvD9D10coding sequence was amplified by PCR using the pscFvD9D10 V_(Hum)plasmid as a template. The sense primer used in this amplificationcarried a unique SpeI restriction site in such a way that the resultingscFvD9D10 sequence could be fused in-frame at the C-terminus of thedimerisation domain.

sense primer:

5′-CGCGACTAGTGCAGAGCGGTAGCGAACTG-3′   (SEQ ID NO 82)

antisense primer:

5′-GCCAGTGAATTCTATTAGTGGTGATG-3′   (SEQ ID NO 83)

The resulting PCR fragment was inserted into the pGEM-T vector andverified by DNA sequence analysis. The resulting plasmid was namedpGEM-TscFvD9D10 f s/e. Subsequently, the MoTABI expressionplasmid wasassembled in a three-point ligation using following fragments: TheN-terminal scFvD9D10 originating from vector pscFvD9D10V_(hum) as aXhoI/EcoRI fragment. This fragment also carried the antibioticresistance gene (Amp), the origin of replication and the expression- andsecretion signals. A second fragment, originating from pGEM-THDH cutwith XhoI and SpeI, carried the helix-turn-helix dimerisation domainalready described previously flanked by human IgG3 upper hinge regions.Finally, a third fragment, originating from the SpeI/EcoRI cutpGEM-TscFvD9D10 f s/e plasmid, carried the C-terminal scFvD9D10 with thehexahistidine tag. The final expressionplasmid was named pMoTAbIH6 (FIG.14) and carried the MoTAbI molecule under control of the lac promotorand the pelB signal sequence as the secretion signal (FIGS. 15 and 16).(SEQ ID NO 84 and 85) To reduce immunogenicity, the hexahistidinesequence was removed using synthetic oligo's in a similar way asdescribed previously for the humanized scFvD9D10, resulting in MoTabI.The MoTAb I expression plasmid was introduced into a suitable E.coliexpression strain, e.g. JM83 and BL21. Good expressionlevels could beobtained in both strains. Detection of the MoTabI molecule (60 kDa) onwestern blot was done with an anti D9D10 rabbit polyclonal antibodyand/or an anti His6 monoclonal antibody (Babco). However, only a minoramount of the MoTAbI molecule was present in a soluble form in thebacterial periplasm. The majority of the MoTAbI molecule was not able totraverse the bacterial membrane and was present as cytoplasmic inclusionbodies. This was confirmed by N-terminal amino acid sequencing whichrevealed still the presence of the pelB signal sequence on the molecule.The functionality of the minor amount of secreted MoTAbI could howeverbe confirmed using an ELISA. In this ELISA, recombinant human IFNγ wascoated onto a polystyreneplate and incubated with periplasmic fractionsoriginating from E.coli cells expressing the MoTAbI molecule. BoundMoTAbI molecules where then detected using a rabbit polyclonal serumgenerated against the D9D10 scFv followed by a peroxidase labeled goatanti rabbit secondary serum.

Since most MoTAbI molecules were present in cytoplasmic inclusionbodies, the molecules were purified from this fraction under denaturingconditions followed by refolding to functional molecules. However, sincethe MoTAbI molecule has the pelB signal sequence still attached, a newcytoplasmic expressionplasmid was constructed. In thisexpressionplasmid, MoTAbI expression is under control of the strongleftward promotor of phage lambda (P_(L)). Since no secretion to theperiplasmic space is necessary, the MoTAbI coding sequence was fuseddirectly to an ATG startcodon. This was accomplished by isolating theMoTAbI coding sequence lacking the pelB signal sequence by PCR from thepMoTAbI expressionplasmid and recloning it into the EcoRV opened pBSK(+)vector (Stratagene). A SapI restriction site giving access to the firstmature codon was hereby generated. After DNA sequence verification theMoTAbI coding sequence was inserted as a SapI blunt/SalI fragment intothe NcoI blunt/SalI cut pIGRI2 vector.

pIGRI2 expressionvector nucleotide sequence

1 TTCCGGGGATCTCTCACCTACCAAACAATGCCCCCCTGCAAAAAATAAAT

51 TCATATAAAAAACATACAGATAACCATCTGCGGTGATAAATTATCTCTGG

101 CGGTGTTGACATAAATACCACTGGCGGTGATACTGAGCACATCAGCAGGA

151 CGCACTGACCACCATGAAGGTGACGCTCTTAAAAATTAAGCCCTGAAGAA

201 GGGCAGGGGTACCAGGAGGTTTAAATCATGGTAAGATCAAGTAGTCAAAA

251 TTCGAGTGACAAGCCTGTAGCCCACGTCGTAGCAAACCACCAAGTGGAGG

301 AGCAGTAACCATGGTTACTGGAGAAGGGGGACCAACTCAGCGCTGAGGTC

351 AATCTGCCCAAGTCTAGAGTCGACCTGCAGCCCAAGCTTGGCTGTTTTGG

401 CGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAA

451 GCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCA

501 CCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAG

551 TGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAA

601 CGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTC

651 GGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGAmTTGAACG

701 TTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACT

751 GCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGC

801 GTTTCTACAAACTCTTTTGTTTATTTTTCTAAATACATTCAAATATGTAT

851 CCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATAAAAGGATCT

901 AGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAG

951 TTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTC

1001 TTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAAC

1051 CACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTT

1101 TTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCT

1151 TCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGC

1201 CTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGC

1251 GATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAA

1301 GGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGG

1351 AGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAA

1401 AGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGG

1451 CAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCT

1501 GGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGA

1551 TTTTmGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAA

1601 CGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT

1651 TCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTT

1701 GAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTC

1751 AGTGAGCGAGGAAGCGGAAGAGCGCTGACTTCCGCGTTTCCAGACTTTAC

1801 GAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGT

1851 TTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCT

1901 GCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGG

1951 AGCACGATCATGCGCACCCGTGGCCAGGACCCAACGCTGCCCGAGATGCG

2001 CCGCGTGCGGCTGCTGGAGATGGCGGACGCGATGGATATGTTCTGCCAAG

2051 GGTTGGTTTGCGCATTCACAGTTCTCCGCAAGAATTGATTGGCTCCAATT

2101 CTTGGAGTGGTGAATCCGTTAGCGAGGTGCCGCCGGCTTCCATTCAGGTC

2151 GAGGTGGCCCGGCTCCATGCACCGCGACGCAACGCGGGGAGGCAGACAAG

2201 GTATAGGGCGGCGCCTACAATCCATGCCAACCCGTTCCATGTGCTCGCCG

2251 AGGCGGCATAAATCGCCGTGACGATCAGCGGTCCAGTGATCGAAGTTAGG

2301 CTGGTAAGAGCCGCGAGCGATCCTTGAAGCTGTCCCTGATGGTCGTCATC

2351 TACCTGCCTGGACAGCATGGCCTGCAACGCGGGCATCCCGATGCCGCCGG

2401 AAGCGAGAAGAATCATAATGGGGAAGGCCATCCAGCCTCGCGTCGCGAAC

2451 GCCAGCAAGACGTAGCCCAGCGCGTCGGCCGCCATGCCGGCGATAATGGC

2501 CTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAG

2551 CGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTC

2601 GCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGG

2651 CACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGA

2701 CGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCT

2751 CTCAAGGGCATCGGTCGGCGCTCTCCCTTATGCGACTCCTGCATTAGGAA

2801 GCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATG

2851 GTGCATGTAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCC

2901 ACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCG

2951 ATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTG

3001 TGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGAATCCACAGG

3051 ACGGGTGTGGTCGCCATGATCGCGTAGTCGATAGTGGCTCCAAGTAGCGA

3101 AGCGAGCAGGACTGGGCGGCGGCCAAAGCGGTCGGACAGTGCTCCGAGAA

3151 CGGGTGCGCATAGAAATTGCATCAACCGCATATAGCGCTAGCAGCACGCCA

3201 TAGTGACTGGCGATGCTGTCGGAATGGACGATATCCCGCAAGAGGCCCGG

3251 CAGTACCGGCATAACCAAGCCTATGCCTACAGCATCCAGGGTGACGGTGC

3301 CGAGGATGACGATGAGCGCATTGTTAGATTTCATACACGGTGCCTGACTG

3351 CGTTAGCAATTTAACTGTGATAAACTACCGCATTAAAGCTAATCGATGAT

3401 AAGCTGTCAAACATGAGAATTAA (SEQ ID NO 86)

The new vector is called pIGRI2MoTAbI. A version lacking thehexahistidine tag was constructed in a similar way starting from theprevious MoTAbI expressionplasmid without hexahistidine tail. The newMoTAbI expressionvectors were subsequently transferred to E.coliexpressionstrains MC1061(pAcI), SG4044(pcI857) and UT5600(pAcI). Asexpected, most of the expressed MoTAbI was present as cytoplasmicinclusionbodies. MoTAbI molecules were purified from cytoplasmicinclusion bodies under denaturing conditions followed by standardrefolding procedures as described by De Bernardez Clark (1998).

4.2. Generation of MoTAb II

The D9D10 MoTAb II is defined as a humanized D9D10 whole antibodymolecule to which a humanized D9D10ScFv sequence was attached at thecarboxyterminus (CH3-domain) of the heavy chain (see FIG. 1). Acomparable type of molecule has already been described in literature(Coloma and Morrison, 1997).

For the expression of the D9D10 MoTAbII protein two fusion genes,respectively coding for heavy and light chain protein of the assembledantibody, were constructed. The heavy chain fusion gene consists of animmunoglobulin leader sequence (D9D 10 V_(K) leader cDNA) followed bythe humanized D9D10 heavy chain variable domain cDNA, a human IgG1 heavychain constant domain (C_(H)1-Hinge-C_(H)2-C_(H)3) cDNA, a short G₃Slinker sequence (Coloma and Morrison, 1997) and the humanized D9D10 ScFvsequence. Alternative linker sequences such as the (G₄ S)₃ sequence orthe flexible and proteolysis-resistant truncated mouse IgG3 upper hingeregion (Pack & Plückthun, 1992) can be used.

The light chain fusion gene is identical to the humanized D9D10recombinant antibody light chain gene (2) and contains the D9D10 V_(K)leader, the humanized light chain variable domain cDNA and the humanIgG1 constant domain (kappa).

Construction of MoTAb II Heavy Chain cDNA

The basic constructs generated for expression of the humanized D9D10antibody could be used as backbone for the MoTAbII constructs. Asdescribed several intermediate cloning constructs, mainly generated byPCR-assembly and -amplification, eventually resulted in two finalconstructs, named pGEMhD9D10_(L) and pGEMhD9D10_(H). The latter plasmidwas used as acceptorfragment after digestion with HindIII and EcoRI,which eliminates the STOP codon for insertion of a HindIII-EcoRIdonorfragment isolated from a plasmid pGEM-T-D9D10HE, resulting in thein frame fusion of the hD9D10_(H) cDNA to a cDNA sequence encoding theGly₃Ser linker followed by the humanizedScFv-module and a STOP codon.The resulting plasmid was named pGEM-MoTAbII_(H).

pGEM-T-D9D10HE was constructed by PCR amplification usingpScFvD9D10V_(hum) as template with primers IG8078 and IG8077. Theresulting 755 bp PCR fragment, containing the Gly₃Ser linker followed bythe humanized scFv-module and a STOP codon, was directly cloned in thepGEM-T vector.

      HindIII IG8078(sense):5′-CCCAAGCTTGGCGGAGGCTCACAGGTGCAGCTGGTGCAGAG-3′     EcoRI (SEQ ID NO 87)IG8077(antisense): 5′-CGGAATTCTACCGTTTGATCTCGAGTTTGG-3′ (SEQ ID NO 88)*Construction of mammalian expression plasmids

Expression in mammalian cell lines was performed completely as describedfor the humanized D9D10 antibody (cf example 2). The cDNA encoding theLdrV_(Hh)C_(H)ScFv or MoTAbII_(H) fusion protein was initially insertedin the pEE6hCMV-BgIII (Lonza biologicals) intermediate expressionvector, under transcriptional control of the hCMV promoter. This wasperformed by transfer of the EcoRI-XbaI DNA insert from pGEMMoTAbIIHinto the pEE6hCMV-BgIII vector. From the pEE6MoTAbII_(H) plasmid acomplete mammalian expression casette, consisting of CMV-promoterfollowed by the fusion gene and a polyadenylation site, was thentransferred as a BgIII/BamHI fragment to the BamHI openedpEE12hD9D10_(L) and pEE14hD9D10_(L) expression plasmids alreadyavailable (construct was earlier described for the humanized D9D10antibody construct in example 2). The final expression plasmids, namedpEE12MoTAbII and pEE14MoTAbII then consisted of the pEE-backbone plasmidcontaining the GS-selection unit, carrying the light chain fusion geneexpression casette followed by a comparable heavy chain fusion geneexpression casette. A schematic representation of both plasmids is givenin FIGS. 17 and 18. The approach of assembling a single expressionplasmid containing separate transcription units for both heavy and lightchains and the selectable marker, is adviced in order to ensurecoamplification with the marker gene. The cDNA sequence encoding thecomplete MoTAbII heavy chain fusion protein is given in FIG. 19 (SEQ IDNO 89). The amino acid sequence of the MoTAbII heavy chain fusionprotein is given in FIG. 20 (SEQ ID NO 90).

Small Scale Expression of D9D10 MoTAbII in COS Cells

Transient expression in COS monkey kidney cells was performed using bothmammalian expression constructs pEE12MoTAbII and pEE14MoTAbII completelyas described for the humanized D9D10 antibody (cf example 2). Qualitycontrol was performed by IFNγ-binding ELISA and SPR-analysis.

ELISA

The same set up was used as described in example 2. Results are shown inFIG. 11. Specific binding to IFNγ is detected. The signal is lower thanthe signal obtained with crude COS supernatant of humanized D9D10.However, no concentrations were determined of MoTAbII.

SPR Analysis

A similar set up was used as described for the evaluation of the murineand humanized scFvD9D10 derivatives. Briefly, murine D9D10 wasimmobilized directly onto a B1 sensorchip at a concentration of 10 μg/mlD9D10 in an acetate buffer pH 4.8 using amine coupling. A fixedconcentration of 8 μg/ml human IFNγ was added, followed by the injectionof either murine D9D10 (10 μg/ml; positive control) or crude COSsupernatant containing MoTAb II. Results are shown in FIG. 21. Thesedata clearly illustrate the presence of active, IFNγ binding moleculesin the COS supernatant. As no exact concentrations were determined ofthe MoTAB II, no affinity data could be calculated.

Inhibition of MHC Class II Induction

cf Example 8.1.

Purification

MoTAbII was purified using classical protein A chromatography (Perry andKirby, 1990; Page and Thorpe, 1996). Quality control of the purifiedconstruct was done by Western Blot (classical technology) and ELISA. Thelatter was performed as described in example 2 and results are shown inFIG. 13. From these results we can conclude that MoTAbII is specificallyinteracting with human IFNγ.

Generation of Stable Mammalian Expression Cell Lines

For generation of stable mammalian expression cell line, two host celllines Ns0 (Galfre and Milstein, 1981; ECACC 85110503) and CHO-K1 (ATCCCCL61) were used. Transfection and selection procedures were completelyidentical as described for the humanized D9D10 whole antibody, using theplasmids pEE12MoTAbII for Ns0 and pEE14MoTAbII for CHO-K1. For both NS0and CHO-K1, several MoTAbII producing cell lines (determined inIFNγ-binding ELISA) were initially isolated and used as parental clonesfor further amplification of recombinant protein expression levels asdescribed earlier.

Production of large amounts of the recombinant protein is performed onbioreactor systems optimal for the respective host cells.

5. Generation of Anti-IFNγ Diabodies

Diabodies are dimeric antibody fragments. In each polypeptide, aheavy-chain variable domain (V_(H)) is linked to a light-chain variabledomain (V_(L)) but unlike scFv's, each antigen-binding site is formed bypairing of one V_(H) and one V_(L) domain from two differentpolypeptides. This is achieved by shortening the linker between theV_(H) and V_(L) domains in each molecule (Holliger et al., 1993). Sincediabodies have two antigen-binding sites they can either be monospecificor bispecific. Monospecific bivalent molecules are generated by theshortening the flexible linker sequence of the scFv molecule to betweenfive and ten residues and by cross-pairing 2 scFv molecules withshortened linker. In order to stabilize the molecule, an optionalcysteine residue can be inserted in the linker. As an example for thedifferent steps involved in such a construction we have documented theconstruction of D9D10 -derived monospecific, humanized anti-IFNγdiabodies. The 15 residue linker of the His6-tagged, humanized scFvD9D10was replaced by the 5 or 10 residue linker using overlap extension PCR.Shortly, both D9D10 V_(H) and V_(L) coding sequences were PCR amplifiedwhereby the V_(H) antisense primer and the V_(L) sense primer havesequences coding for the 5- or 10-mer linker sequence. The resultingV_(H) and V_(L) PCR fragments were subsequently mixed and a second PCRwith the V_(H) sense and V_(L) antisense primers was performed. Theresulting PCR fragment is cloned into the pBSK(+) plasmid (Stratagene)en verified by DNA sequence analysis (FIGS. 22-25) (SEQ ID NO 91-94).The D9D10 diabody coding sequence was subsequently transferred as a SapIblunt/EcoRI fragment and inserted into the NcoI blunt/EcoRI openedvector pTrc99A (Amann et al., 1988). In this vector, expression of thediabodies is under control of the IPTG inducible Trc promotor. Thediabodies were expressed in E. coli strains HB101 or JM83. Periplasmicfractions were prepared following a modified protocol described by Neuand Heppel (1965). Briefly, cells were harvested by centrifugation andresuspended in ice cold shockbuffer (100 mM Tris-HCl pH 7.4; 20%sucrose; 1 mM EDTA pH8). After incubation on ice during 10 min. withoccasional stirring, the mixture was centrifuged at 10.000 rpm during1.5 min. The supernatans was removed and the pellet was immediatelyresuspended in ice cold distilled water. After incubation on ice during10 min. with occasional stirring, the mixture was centrifuged at 14.000rpm and the obtained supernatans was the soluble periplasmic fraction.The periplasmic fractions were tested for binding to IFNγ usingSPR-analysis. The experimental set up was as described in example 2. Theundiluted samples were injected onto the surface of a B1 sensorchipcoated with murine D9D10 onto which IFNγ was injected. Results obtainedwith L5 D9D10 diabodies are shown in FIG. 26. A clear, specific bindingof the diabodies was detected. Comparable results were obtained with theL10 D9D10 diabody.

The bivalent, monospecific diabody molecules are purified from theperiplasmic extract via IMAC or from periplasmic inclusion bodies usingdenaturing conditions followed by refolding.

Overlap Extension PCR Primers for the L10D9D10 Diabodies:

D9D10V_(H) forward (sense) primer

5′GGCCGCTCTTCGAAATACCTATTGCCTACGGCAG3′  (SEQ ID NO 95)

D9D10L10V_(H) backward (antisense) primer

5′-CTGGGTCAGTACGATGTCAGAGCCACCTCCGCCTGAACCGCCTCCACCTGAGGAGACGGTGACCGTGGTC-3′  (SEQ ID NO 96)

D9D10L10V_(L) forward (sense) primer

5′-GTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGACATCGTACTGACCCAGAGCC-3′  (SEQ ID NO 97)

D9D10V_(L) backward (antisense) primer

5′-GCCAGTGAATTCTATTAGTGGTGATG-3′   (SEQ ID NO 98)

Overlap Extension PCR Primers for the L5 D9D10 Diabodies:

D9D10V_(H) forward (sense) primer

5′-GGCCGCTCTTCGAAATACCTATTGCCTACGGCAG-3′   (SEQ ID NO 95)

D9D10L5V_(H) backward (antisense) primer

5′-CTGGGTCAGTACGATGTCTGAACCGCCTCCACCTGAGGAGACGGTGACCGTGGTC-3′   (SEQ IDNO 99)

D9D10L5V_(L) forward (sense) primer

5′-GTCACCGTCTCCTCAGGTGGAGGCGGTTCAGACATCGTACTGACCCAGAGCC-3′   (SEQ ID NO100)

D9D10V_(L) backward (antisense) primer

5′-GCCAGTGAATTCTATTAGTGGTGATG-3′   (SEQ ID NO 98)

6. Generation of Anti-IFNγ Triabodies

The construction of triabody molecules was analogous to the schemedescribed above for diabody molecules, except that the (G₄S)₃ linkerbetween the humanized D9D10 VH and VL was completely deleted (FIGS. 27and 28) ( SEQ ID NO 101-102) (zero-residue linker or −1-residue linkeraccording to the Kabat numbering (Kortt et al., 1997; Iliades et al.,1997) ). The humanized D9D10 triabody construct is a mono-specificmolecule resulting from the spontaneous association of threezero-residue linker (or −1-residue) D9D10 scFv molecules in thebacterial periplasm. A trimer was formed whereby three pairs of V_(H)and V_(L) domains interact to form three active antigen combining sites.If necessary, in order to drive triabody formation as well as tomaintain stability, we can explore the possibility of introducingadditional association domains or disulfide bridges.

The produced triabodies were tested for IFNγ binding using SPR-analysis.Periplasmic fractions were prepared as described in example 5.SPR-analysis was performed as described in example 5. Results are shownin FIG. 29. A clear, specific binding of the triabody was obtained.

The triabody molecules were purified from the periplasmic extract, madefrom uninduced bacterial cultures, via IMAC and further by gelfiltration or alternatively by purification under denaturing conditionsfrom periplasmic inclusionbodies followed by refolding. The multimericbehaviour of the purified molecules was analysed. The ability of thepurified triabody to bind human interferon γ was tested usingSPR-analysis and ELISA experiments as described earlier. For these testswe produced milligram amounts of highly purified material in a suitableE.coli expression system.

Overlap Extension PCR Primers for the L0 D9D10 Triabodies:

D9D10V_(H) forward (sense) primer

5′-GGCCGCTCTTCGAAATACCTATTGCCTACGGCAG-3′   (SEQ ID NO 95)

D9D10L0V_(H), backward (antisense) primer

5′-CTGGGTCAGTACGATGTCTGAGGAGACGGTGACCGTGGTC-3′   (SEQ ID NO 103)

D9D10L0V_(L) forward (sense) primer

5′-GTCACCGTCTCCTCAGACATCGTACTGACCCAGAGCC-3′   (SEQ ID NO 104)

D9D10V_(L) backward (antisense) primer

5′-GCCAGTGAATTCTATTAGTGGTGATG-3′   (SEQ ID NO 98)

7. Generation of MoTAb's (and BiTAb's) Originating from Fusion Proteins,from Serum Multisubunit Proteins and from scFv's

The multi subunit (oligomeric) structure of proteins may be exploited toobtain multivalent antibodies, when they are used as fusion partner withscFv antibodies. Either the whole polypeptide chain, or the associationsequence domain may be used as fusion partner.

For example, haemoglobin is a tetrameric serum protein, consisting from2 alpha and 2 beta globin subunits. The dimer dissociation constant isestimated to be in the order of 1 nM (Pin et al., 1990). Thetetramer—dimer dissociation constant of haemoglobin in oxy-conformationwas studied by gel filtration on Superose 12 and was calculated to be 1μM (Manning et al, 1996). Although non-covalent associations are knownto be susceptible to equilibrium rules, it has been described that thesubunit interactions are favoured in concentrated protein solutions likeserum and also may be increased by the presence of other stabilisingcompounds (Srere and Mathews, 1990).

Recombinant haemoglobin expression has been extensively investigated asa possible blood substitute in order to circumvent the transmission ofinfectious disease agents during blood transfusion. The alpha- andbeta-globin polypeptides have already been expressed from a singleoperon in E. coil (Hoffman et al., 1990). In this case, the recombinanthaemoglobin was purified from the soluble cytoplasmatic fraction and thetetrameric E. coli product had essentially the same characteristics asthe native protein. Analogous results were obtained when recombinanthaemoglobin was expressed in S. cerevisiae (Pagnier et al., 1992; Mouldet al., 1994; Sutherland-Smith et al., 1998).

Protein engineering strategies (Olson et al., 1997) and chemicalmodification by pegylation (Pettit and Gombotz, 1998) are investigatedto enhance the stability and the circulation half times in vivo. Sofusion of relevant scFv molecules to the respective alpha and betasubunit of human haemoglobin and expression of the fusion proteins froma single operon in either E. coli or S. cerevisiae would yield afunctional tetrameric monospecific (if identical scFv's are used) orbispecific (when different scFv's are used) molecules at high level.

8. Evaluation of Anti-IFNγ Neutralizing Molecules

8.1. Inhibition of MHCII-induction

In the first experiments, the effect of IFNγ on the induction of MHCclass II expression on human keratinocytes was examined. For this,primary human keratinocytes (passage 1) were cultured with twoconcentrations of human IFNγ (100 U/ml and 200 U/ml) during 24 and 48hours. After culture, cells were collected and the expression of MHCclass II antigen on the activated keratinocytes was measured byFACS-scan after staining (30 minutes at 4° C.) of the cells with aPE-labelled anti-MHC-class II mAb. The results showed that restingkeratinocytes do not express MHC class II molecules and that IFNγinducesthe expression after 24 hours in a dose-dependent way. The induction isstill enhanced after 48 hours of culture.

In the next study, the effect of anti-human IFNγD9D10H3 full sizeantibody or scFvD9D10-cmyc on the IFNγ-induced MHC-Class II expressionon human keratinocytes was examined. In this experiment, human primarykeratinocytes (passage 1) were cultured with human IFNγ (100 U/ml) inthe presence or absence of different concentrations (2-0.5-0.12-0.03)D9D10 Ab or D9D10scFv for 48 hours. IFNγ was preincubated with D9D10H3or scFvD9D10 during 1 hour at 37° C. before adding to the keratinocytes.After culture, cells were collected and the expression of MHC-Class IIon these activated keratinocytes was measured. For this, keratinocyteswere incubated (30 minutes at 4° C.) with a PE-labelled anti-MHC-ClassIImAb (Becton Dickinson), washed twice with PBS and fixed. The MHC-ClassII expression was further analysed on a FACS-scan. The results of theseexperiments are represented in FIG. 30. It is shown that the MHC classII antigen is not expressed on the membrane of resting keratinocytes andthat IFNγ clearly induces this MHC class II expression. This IFNγinduced MHC class II expression is dose dependently inhibited by D9D10H3and to a lesser extent by scFvD9D10. We can conclude that about 4 timesmore scFv (0.12 μg/ml) than full size antibody (0.5 μg/ml) is needed toobtain a 50% inhibition of the IFNγ-induced MHC class II expression onkeratinocytes.

Similar experiments were performed in order to evaluate theneutralization capacity of humanized D9D10 and MoTAbII. Results aresummarized in FIG. 31. Although in this experiment, MHC class IIinduction could be only induced to a lesser extent, both humanized D9D10and MoTAbII clearly inhibit the IFNγ-induction.

8.2. Inhibition of Anti-viral Activity

For neutralization of the antiviral activity of hIFNγ, serial dilutionsof samples (anti-IFNγ constructs) were prepared in microtiter plates. Toeach well, hIFNγ was added to a final concentration of 5 antiviralprotection Units/ml, as tested on A549 cells. The mixtures wereincubated for 4 h at 37° C. and 25000 A549 cells were added to eachwell. After an incubation period of 24 at 37° C. in a CO₂ incubator, 25μl of 8×10⁵ PFU EMC virus/ml was added to the cultures for at least 24h. As soon as virus-infected control cultures reached 100% celldestruction, a crystal violet staining was performed in order toquantify surviving cells. The neutralization capacity of the anti-IFNγconstructs was defined by the concentration of the construct needed toneutralize 95% of the antiviral activity of 5U/ml human IFNγ. Theneutralization potency of the scFvD9D10 and the humanized scFvD9D10 wasdetermined and was 1.2 μg/ml and 1.5 μg/ml, respectively.

8.3. Beneficial Effects in Septic Shock in Mice

Septic shock has been demonstrated to be a complex human diseasemanifestation that occurs after the release of lipopolysaccharide (LPS)into the circulation. The subsequent production of high cytokine levelsin the serum are known to play a crucial role in septic shock. Wegenerated data in a mouse model system using an anti-mouse IFNγ calledF3 (Froyen et al., 1995).

The generalized Shwartzman model is a lethal shock syndrome inexperimental animals which is elicited by 2 consecutive injections ofLPS. In the laboratory of prof. Billiau (Rega Institute, CatholicUniversity Leuven, Belgium), such a model was developed in mice (Billiauet al., 1987). At time 0, the mice were injected with 5 μg LPS into thefootpad, followed 24 h later by a second intravenous injection of 100μg. Morbidity and mortality was scored for 5 days. Untreated animalsnormally died within 2 days after the second injection. Mice pretreatedwith the anti-muIFNγ antibody F3 were completely protected against thelethal effect and only showed moderate disease symptoms. This protectioncould be achieved with as little as 2.4 μg F3 given 24 h before thefirst injection. In order to score the severity of the disease, thesymptoms were classified in 5 groups:

Score 0: not sick or mild piloerection

Score 1: piloerection and diarrhoea

Score 2: hemorhagic conjunctivitis and bleeding at the mouth and anus

Score 3: paralysis of the hind legs

Score 4: death

The highest score that could be obtained is 4. Since the number of micein each group was relatively low (5), we established a limit of thedisease score (=2) that had to be reached in the saline group in orderto be a representative experiment.

The schedule we used in order to compare F3 and its scFv in thisShwartzman model was as follows: NMRI mice were given the preparativedose of 5 μg LPS at time 0. At the time points +6 h, +12 h and +23 h themice were injected ip with 190 μg scFvF3 (Froyen et al., 1995) or 30 gF3. Control animals were given saline at the same time points. Eachgroup consisted of 5 mice. The mice were given a score according to theabove mentioned classification.

In the first experiment, 40% more mice were protected in the scFvF3group when compared with the control group. A second experiment was setup using a slightly adapted protocol: an additional injection was givenat timepoint +3 h. The result of this experiment (shown in table) wassimilar to that of experiment 1 in that 40% more mice survived in thescFvF3 group in comparison with the control group as can be seen in FIG.32. In addition to scFvF3, a Fab antibody fragment of F3 was included inthe second group. All these mice survived the experiment.

The mean disease scores of these experiments, demonstrate a significantdifference for both F3 and the scFv compared to the control group. Themean disease scores of the 5 mice of each group were as follows:

Saline scFvF 3F3 FabF3 exp. 1 3.2 1.8 0.0 ND exp. 2 2.6 0.8 0.6 0.6

8.4. Beneficial Effects During Cachexia in Mice

In a model for cachexia developed at the Rega Institute (Matthys et al.,1991), nude mice were injected intraperitoneally (ip) with CHO cellsproducing mouse IFNγ (Mick cells). Mice receiving CHO-Mick cells willexhibit cachexia (including body weight loss) within 48 hours. Thecachectic effect is correlated with the number of Mick cells. Thus withsmall tumor cell inocula (0.8-3.0×10⁷ cells), cachexia is transient andmice will completely recover. However, with high inocula (>3.4×10⁷cells), mice continue to loose weight and will die within 7 days. It isshown that IFNγ plays an essential role in the pathogenesis of theMick-induced cachexia as monoclonals against IFNγ can reverse thewasting effect: pretreatment (day −1) with the anti-muIFNγ antibody F3inhibits cachexia.

In order to compare the effects of F3 and its scFv on the establishedcachexia model, the following experiment has been set up: mice wereinjected with 2-4×10⁷ Mick cells on day 0 and antibody preparations wereadministered ip at time points +1.5 h, +6 h, +22 h and +66 h relative tothe time of Mick cell inoculation. For scFvF3, a dose of 190 μg wasgiven each injection while for F3, 40 μg was given. Control animals wereinjected with saline at the same time points. In each group, 3 or 4 micewere used. Mice were weighed for 10 consecutive days and mortality wasscored. The results of 2 independent experiments are shown in FIG. 33.The mice treated with scFvF3 were better protected against the cachecticeffect than the control mice.

These results also indicate that scFvF3 antibody fragments do have aprotective effect of cachexia but to a lesser extent than the parentalF3 antibody. Although results were promising, it was clear that theeffect of the scFv fragment was limited either due to its fast clearanceor to lowered affinity. Optimization of the injection schedule wasneeded to obtain comparable results.

8.5. Beneficial Effects in Septic Shock in Non-human Primates

The best documented sepsis model in non-human primates is the one inwhich baboons are given lethal infusions of E.coli. As described byCreasey et al. (1991), response to lethal E.coli challenge occurs in 3stages: an inflammatory stage marked by a fall in white blood cell count(0-2 hr) and the appearance in plasma of TNFα, IL-1β and IL-6; acoagulant stage marked by a fall in fibrinogen concentration (2-6 hr);and a hypoxic cell injury stage marked by a rise in SGPT/BUN and by agradual cardiovascular collapse, and death (6-24 hr).

Since the baboon animal model was not readily available, we areestablishing a comparable rhesus monkey model. D9D10 and derivedconstructs interacted well with rhesus IFNγ as determined in anantiviral bioassay (set up as described in example 7.2.).

Septic shock can be induced by infusion either of life bacteria or ofendotoxin in sedated monkeys. After administration of differentconcentrations of the D9D10 anti-hIFNγ derivatives, several parametersare monitored including:

mortality (should be 100% in control (non-treated) group)

pathophysiology

serum concentration of cytokines such as TNFα, IL-1 and IL-6 using ELISAor bioassay (Villinger et al., 1993)

endotoxin profile using the limulus amoebocyte lysate assay

8.6. Beneficial Effects During Experimental Autoimmune Encephalomyelitisin Non-human Primates

A. Pharmacokinetics of D9D10 and derivatives in monkey and effect onhIFNγ clearance

The clearance of the antibody derivatives is of importance as moleculeswith a slow clearance have a prolonged efficacy. This implicates thatless material has to be injected which is better for the patient andwhich is cost effective, especially when a longer treatment period isadvisable. Therefore, complexes of IFNγ and D9D10 derivatives are usedin clearance studies in non-human primates as a prerequisite to guidefurther in vivo studies in these animals.

The clearance of D9D10, scFvD9D10H6⁻, D9D10 MOTAB I and D9D10 MOTAB II,is monitored after a bolus injection in healthy marmoset. SpecificELISA's are used for monitoring; no labelling of the antibody constructsis required.

Blood clearance of radiolabelled marmoset IFNγ after a bolus intravenousinjection alone or in combination with one of the antibody constructsare also performed.

B. Beneficial Effects of the D9D10 Antibody Constructs on EAE inNon-human Primates

In order to evaluate the therapeutic potential of the anti-IFNγ MabD9D10 and derivatives, we are testing this antibody in a relevantnon-human primate model for MS as the final step in our preclinicalresearch. This model is required since the antibody is notcross-reacting with IFNγ from rodents and the biological activity ofIFNγ is very species specific (huIFNγ is not active on cells other thanhuman or non-human primates (Terrell and Green, 1993)). D9D10 andderived constructs interact well with marmoset IFNγ as determined in anantiviral bioassay (set up as described in example 7.2.) and usingsurface plasmon resonance (set up as described in example 1).

The EAE model is chosen as it is a generally accepted model for MultipleSclerosis. We opt for the EAE model in common marmoset (Callithrixjacchus) as it is well developed (Massacesi et al., 1995; Genain et al.,1995), it has a pathology of MR-detectable lesions which reflects thosein MS and the model shows a high incidence of EAE induction with achronic progressive/relapsing-remitting course.

Acute PK-Tox

A limited PK-Tox study required by ethical prescriptions in all researchinvolving non-human primates, is set up in order to test the toxicity ofthe substances administered either intravenously or in the lumbarcerebrospinal fluid (CSF), as the contribution of systemic and/or localIFNγ to the development of the disease is still unclear. Relatively highconcentrations of the antibody preparations, especially for the scFv,are injected intravenously as one of our goals is to reach therapeuticalconcentrations in the CNS. Although it is known that BBB leakage occursat the site of inflammation (‘t Hart, personal communication ), apositive concentration gradient will be beneficial.

Timing of the Study

Determination of the baseline parameters is done 1 week prior toadministration of the study drug. Animals are observed for signs oftoxicity for 30 days. During this period pharmacokinetic parameters aremonitored. Six weeks after the administration of the study drug anadditional blood sample is collected to determine whether or not theanimals mounted an immune response to any of the D9D10 constructs or torecombinant marmoset IFNγ.

Parameters

During This Study the Following Parameters are Determined

Clinical Monitoring

Daily Food consumption Weekly Body weights Day 14, 28 HaematologyClinical chemistry Urine analysis

Immunological Monitoring

Serum and CSF levels of humanized D9D10, MoTAbI and II or IFNγ aremeasured at different time points. When severe toxicity occurs in one ofthe animals, the animal are sacrificed and subjected to a detailednecropsy, in order to determine whether this toxicity is drug related.

Diffusion of D9D10 Derivatives into the Lesions

As both systemically and locally (in the brain) produced IFNγ can have adisease promoting role in EAE, antibody derivatives must be able toneutralize both. Consequently, transudation of the D9D10 derivativesinto the lesions in brain and spinal cord is necessary for a localeffect on IFNγ. However, it is known that in MS the blood brain barrieris impaired in a subset of the active brain lesions for a limited periodof time. More specifically, BBB breakdown is reflecting the state ofinflammation (Hawkins et al., 1990).

The differential ability of the anti-IFNγ constructs to enter the brainis crucial for the choice of the component(s) which will be used forevaluation of the therapeutic efficacy of an anti-IFNγ treatment in EAE.

The entry of the constructs into the brain compartment is measured bypost-mortem magnetic resonance imaging (MRI)-scan of the brain and thespinal cord of a relapsing monkey, injected intravenously with agadolinium-diethylene-triamine pentaacetic acid (Gd-DTPA)-labelled D9D10construct 1 hour prior to sacrifice. MRI-scans are compared and arerelated to an MRI-scan taken just before death after an injection with asmall gadolinium salt that easily enters through leakages in the BBB(Gonzalez-Scarano et al., 1987; Hawkins et al., 1990; Youl et al, 1991).

These results reveal which D9D10 construct most easily enters the brainand which molecule eventually enters the active lesions where the BBB isalready restituted.

Therapeutic Treatment of Marmoset Monkeys Undergoing EAE Disease Relapse

The therapeutic effect of either systemically or locally administeredanti-IFNγ on the outcome of EAE in marmoset is evaluated. The start ofthe treatment of the monkeys is situated at the beginning of the firstrelapse of EAE, which usually occurs several months after the initialimmunization. During the experiment the following observations, analysisand measurements are carried out as of the time of relapse.

Clinical Monitoring

The severity of EAE is scored daily on an arbitrary scale modified fromMassacesi et al. (1 995)

Body weight and body temperature (at time of blood sampling)

Behavioural tests for monitoring the failure of neurological functions

Magnetic resonance imaging (MRI) of the CNS

Biochemical parameters: neopterin (specifically formed in activatedmacrophages) is measured in urine

Immunological Monitoring

At several indicated time points serum is taken to monitor the bloodlevels of the antibody constructs or IFNγ and to monitor the marmosetanti-mouse or anti-IFN response.

Pathology

MRI-guided histopathology analysis has proven a powerful tool fordetailed analysis of MR-detectable lesions with histological methods.Briefly, at a chosen moment but preferably shortly after in vivoMR-images have been recorded, the monkey are euthanised. The brain andspinal cord is carefully excised and fixed in toto for 3 days in 4%buffered formaldehyde. Then a T2-weighted scan is made in axial andcoronal direction, with a slice thickness of 1 mm covering the wholebrain. For orientation of the axial slices of in vivo and in vitroimages the anterior and posterior tips of the corpus callosum are usedas internal reference points.

The excellent structural conservation and the high resolution of theMR-image make accurate three-dimensional localisation of potentiallesions possible. Regions of interest are subsequently excised andhistologically analysed for infiltrating cells (Haematoxylin-eosin),demyelisation (KLB staining of myelin lipids) and axonal structure(silver impregnation acc. to Boielschowsky).

One half of an excised brain and spinal cord is snap-frozen in liquidnitrogen. Thin cryosections are made and processed for immunohistologystaining, such as for visualisation of cytokine secreting cells(especially IFNγ) or for phenotyping of infiltrated or tissue cells.

8.7. Beneficial Effects of Anti-IFNγ Antibody Constructs in Crohn'sDisease

A. In vitro Assay Using Patient-derived Lymphocytes and AntigenPresenting Cells

Lymphocytes isolated from either peripheral blood or surgical specimen(lamina propria or ileum E) from patients with Crohn's disease, are usedfor assessment of cytokine profile, lymphotyping, and functionalcytotoxicity. The latter is performed by adding patient-derived antigenpresenting cells and measuring the cytokine profile. The effect ofanti-IFNγ derived antibody constructs on cytokine production ismeasured.

B Anti-IFNγ Treatment of Crohn's Disease

Patients with active Crohn's disease are infused with anti-IFNγ in adose ranging from 1 to 20 mg/kg. Responders in the study may continue toreceive repeated doses of anti-IFNγ. In all patients, clinical responsesare observed and Crohn's disease activity index (CDAI) is determined.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

Allen S. J., Baker D., O'Neill J. K., Davison A. N. and J. L. Turk(1993) Isolation and characterization of cells infiltrating the spinalcord during the course of chronic relapsing experimental allergicencephalomyelitis in the Biozzi AB/H mouse. Cell. Immunol. 146: 335-350.

Amann E., Ochs B. & Abel K. J. (1988) Tightly regulated tac promotervectors useful for the expression of unfused and fused proteins inEscherichia coli, Gene, 69: 301-315.

Arenberger P., Ruzicka T. and L. Kemeny (1991) Effect of cyclosporin onepidermal 12(S)-hydroxyeicosatetraenoic acid binding sites. SkinPharmacol. 4:272-277.

Asadullah K., Renz H., Docke W. D., Otterbach H., Wahn U., Kottgen E.,Volk H. D. and W. Sterry (1997) Verrucosis of hands and feet in apatient with combined immune deficiency. J. Am. Acad. Dermatol.36:850-852.

Barker J. N., Goodlad J. R., Ross E. L., Yu C. C., Groves R. W. and D.M. MacDonald (1993) Increased epidermal cell proliferation in normalhuman skin in vivo following local administration of interferon-gamma.Am. J. Pathol. 142: 1091-1097.

Bebbington C. R., Renner G., Thomson S., King D., Abrams D. And G. T.Yarranton (1992) High-level expression of a recombinant antibody frommyeloma cells using a glutamine synthetase gene as an amplifiableselectable marker. Biotechnology 10:169-75.

Bienvenu J., Doche C., Gutowski M. C., Lenoble M., Lepape A. and J. P.Perdrix (1995) Production of proinflammatory cytokines involved in theTH1/TH2 balance is modulated by pentoxifylline. J. Cardiovasc.Pharmacol. 25: S80-S84.

Billiau A. (1996) Interferon-γ: biology and role in pathogenesis.Advances in Immunology 62: 61-130.

Billiau A., Heremans H., Vandekerckhove F. and C. Dillen (1987)Anti-interferon-gamma antibody protects mice against the generalizedShwartzman reaction. Eur. J. Immunol. 17: 1851-1854.

Billiau A., Heremans H., Vandekerckhove F., Dijkmans R., Sobis H.,Meulepas E. and H. Carton (1988) Enhancement of experimental allergicencephalomyelitis in mice by antibodies against IFN-γ. J. Immunol. 140:1506-1510.

Boissier M-C, Chiocchia G., Bessis N., Hajnal J., Garotta G., NicolettiF. and C. Fournier (1995) Biphasic effect of interferon-γ in murinecollagen-induced arthritis. Eur. J. Immunol. 25: 1184-1190.

Bone R. C. (1992) Toward an epidemiology and natural history of SIRS(systemic inflammatory response syndrome). JAMA 101: 1481-1483.

Brown R. R., Ozaki Y., Datta S. P., Borden E. C., Sondel P. M. and D. G.Malone (1991) Implications of interferon-induced tryptophan catabolismin cancer, auto-immune diseases and AIDS. Adv. Exp. Med. Biol.294:425-435.

Bucklin S. E., Russell S. W. and D. C. Morrison (1994) Participation ofIFN-γ in the pathogenesis of LPS lethality. Bacterial Endotoxins: BasisScience to Anti-Sepsis Strategies, pp. 399-406, Wiley-Liss.

Casey J. L., Keep P. A., Chester K. A., Robson L., Hawkins R. E., and R.H. J. Begent (1995) Purification of bacterially expressed single chainFv antibodies for clinical applications using metal chelatechromatography. J. Immunol. Methods 179: 105-116.

Chan L. S. and K. D. Cooper (1994) A novel immune-mediated subepidermalbullous dermatosis characterized by IgG autoantibodies to a lower laminalucida component. Arch. Dermatol. 130:343-347.

Chomczynski P. And Sacchi N. (1987) Single-step method of RNA isolationby acid guanidinium thiocyanate-phenol-chloroform extraction. Anal.Biochem. 162: 156-159.

Cockett M. I., Bebbington C. R. and G. T. Yarranton (1990) High levelexpression of tissue inhibitor of metalloproteinases in Chinese hamsterovary cells using glutamine synthetase gene amplification. Biotechnology8: 662-7.

Coloma M. J. and S. L. Morrison (1997) Design and production of noveltetravalent bispecific antibodies. Nature Biotech. 15: 159-163.

Courtney L. P., Phelps J. L. and L. M. Karavodin (1994) An anti-Il-2antibody increases serum halflife and improves anti-tumor efficacy ofhuman recombinant interleukin-2. Immunopharmacol. 28: 223-232.

Creasey A. A., Stevens P., Kenney J., Allison A. C., Warren K., CatlettR., Hinshaw L. and F. B Taylor Jr (1991) Endotoxin and cytokine profilein plasma of baboons challenged with lethal and sublethal Escherichiacoli. Circ. Shock. 33: 84-91.

De Bernardez Clark E. (1998) Refolding of recombinant proteins. CurrentOpinion in Biotechnology 9: 157-163.

de Kruif J. and T. Logtenberg (1996) Leucine zipper dimerised bivalentand bispecific scFv antibodies from a semi-synthetic antibody phagedisplay library. J. Biol. Chem. 271: 7630-7634.

Denz H., Orth B., Weiss G., Herrmann R., Huber P., Wachter H. and D.Fuchs (1993) Weight loss in patients with hematological neoplasias isassociated with immune system stimulation. Clin. Investig. 71:37-41.

Desmet J., De Maeyer M., Hazes B. And I. Lasters (1992) The Dead EndElimination Theorem and its use in protein side chain positioning.Nature 356: 539-542.

de St. Groth F. and D. Scheidegger (1980) Production of monoclonalantibodies: strategy and tactics. J. Immunol. Methods 35:1-21.

Doherty G. M., Lange J. R., Langstein H. N., Alexander H. R., Buresh C.M. and J. A. Norton (1992) Evidence for IFNγ as a mediator of thelethality of endotoxin and tumor necrosis factor-α. J. Immunol. 149:1666-1670.

Duong T. T., Finkelman F. D., Singh B. and G. H. Strejan (1994) Effectof anti-interferon-γ monoclonal antibody treatment on the development ofexperimental allergic encephalomyelitis in resistant mouse strains. J.Neuroimmunol. 53: 101-107.

Duprez E., Tong J-H, Dérré J., Chen S-J, Berger R., Chen Z. And LanotteM. (1997) JEM-1, a novel gene encoding a leucine-zipper nuclear factorupregulated during retinoid-induced maturation of NB4 promyelocyticleukaemia. Oncogene 14: 1563-1570.

Dustin M. L., Singer K. H., Tuck D. T. and T. A. Springer (1988)Adhesion of T lymphoblasts to epidermal keratinocytes is regulated byinterferon-γ and is mediated by intercellular adhesion molecule 1(ICAM-1). J. Exp. Med. 167: 1323-1340.

Fanger M. W., Morganelli P. M. and P. M. Guyre (1992) Bispecificantibodies. Crit. Rev. Immunol. 12:101.

Fisher C. J. Jr, Agosti J. M., Opal S. M., Lowry S. F., Balk R. A.,Sadoff J. C., Abraham E., Schein R. M. and E. Benjamin (1996) Treatmentof septic shock with the tumor necrosis factor receptor:Fc fusionprotein. The Soluble TNF Receptor Sepsis Study Group. N. Engl. J. Med.334: 1697-1702.

Freedman A. S., Freeman G. J., Rhynhart K. and L. M. Nadler (1991)Selective induction of B7/BB-1 on interferon-gamma stimulated monocytes:a potential mechanism for amplification of T cell activation through theCD28 pathway. Cell. Immunol. 137: 429-437.

Froyen G., Ronsse I. and A. Billiau (1993) Bacterial expression of asingle-chain antibody fragment (SCFV) that neutralizes the biologicalactivity of human interferon-γ. Mol. Immunol. 30:805-812.

Froyen G., Billiau A., Buyse M.-A. and De Waele P. (1995) The expressionof a ScFv antibody fragment against IFN-gamma. Med. Fac. Landbouww.Univ. Gent, 60/4a.

Galfre G.and C. Milstein (1981) Preparation of monoclonal antibodies:strategies and procedures. Methods-Enzymol. 73: 3-46.

Genain C. P., Nguyen M.-H., Letvin N. L., Pearl R., Davis R. L., AdelmanM., Lees M. B., Linington C. and S. L. Hauser (1995a) Antibodyfacilitation of multiple sclerosis-like lesions in a nonhuman primate.J. Clin. Invest. 96: 2966-2974.

Genain C. P., Roberts T., Davis R. L., Nguyen M. H., Uccelli A., FauldsD., Li Y., Hedgpeth J. and S. L. Hauser (1995b) Prevention of autoimmunedemyelination in non-human primates by a cAMP-specific phosphodiesteraseinhibitor. Proc. Natl. Acad. Sci. USA 92: 3601-3605.

Ghetie, M. A. and E. S. Vitetta (1994) Recent developments inimmunotoxin therapy. Curr. Opin. Immunol. 6:707.

Gluzman Y. (1981) SV40-transformed simian cells support the replicationof early SV40 mutants. Cell 23: 175-82.

Gonzalez-Scarano F., Grossman R. I., Galetta S., Atlas S. W. and D. H.Silberberg (1987) Multiple sclerosis disease activity correlates withgadolinium-enhanced magnetic resonance imaging. Ann. Neurol. 21:300-306.

Gordon E. J., Myers K. J., Dougherty J. P., Rosen H. and Y. Ron (1995)Both anti-CD11a (LFA-1) and anti-CD11b (MAC-1) therapy delay the onsetand diminish the severity of experimental autoimmune encephalomyelitis.J. Neuroimmunol. 62: 153-160.

Gorczynski, R. M. (1995) Regulation of IFN-gamma and IL-10 synthesis invivo, as well as continuous antigen exposure, is associated withtolerance to murine skin allografts. Cell. Immunol. 160:224-231.

Gottlieb S. L., Gilleaudeau P., Johnson R., Estes L., Woodworth T. G.,Gottlieb A. B. and J. G. Krueger (1995) Response of psoriasis to alymphocyte-selective toxin (DAB₃₈₉IL-2) suggests a primary immune, butnot keratinocyte, pathogenic basis. Nature Medicine 1:442.

Griffiths C. E. M., Powles A. V., Leonard J. N., Fry L., Baker B. S. andH. Valdimarsson (1986) Br. Med. J. 293:731-732.

Hartung H. P., Schafer B., Van Der Meide P. H., Fierz W., Heininger K.and K. V. Toyka (1990) The role of interferon-gamma in the pathogenesisof experimental autoimmune disease of the peripheral nervous system.Annal Neurol. 27: 247-257.

Hawkins C. P., Munro P. M. G. and K.Mackenzie (1990) Duration andselectivity of blood-brain barrier breakdown in chronic relapsingexperimental allergic encephalomyelitis studied by gadolinium-DTPA andprotein markers. Brain 113: 365-378.

Heremans H., Dillen C., Groenen M., Martens E. and A. Billiau (1996)Chronic relapsing experimental autoimmune encephalomyelitis (CREAE) inmice: enhancement by monoclonal antibodies against interferon-gamma.Eur. J. Immunol. 26: 2393-2398.

Hoffman S. J., Looker D. L., Roehrich J. M., Cozart P. E., Durfee S. L.,Tedesco J. L. and G. L. Stetler (1990) Expression of fully functionaltetrameric human hemoglobin in Escherichia coli Proc. Natl. Acad. Sci.USA 87: 8521-8525.

Holliger P., Prospero T. and G. Winter (1993) “Diabodies”: smallbivalent and bispecific antibody fragments. Proc. Natl. Acad. Sci. USA90:6444.

Hurle M. R. and M. Gross (1994) Protein engineering techniques forantibody humanization. Curr. Opin. Biotech. 5:428-433.

Huynh H. K., Oger J. and K. Dorovini-Zis (1995) Interferon-betadownregulates interferon-gamma-induced class II MHC molecule expressionand morphological changes in primary cultures of human microvesselendothelial cells. J. Neuroimmunol. 60: 63-73.

Iliades P., Kortt A. A. and P. J. Hudson (1997) Triabodies: single chainFv fragments without a linker form trivalent trimers. FEBS Lett409:437-441.

Ito W. and Y. Kurosawa (1993) Development of an artificial system withmultiple valency using an Fv fragment fused to a fragment of protein A.J. Biol. Chem. 268:20668.

Iwagaki H., Hizuta A., Tanaka N. and K. Orita (1995) Plasmaneopterin/C-reactive protein ratio as an adjunct to the assessment ofinfection and cancer cachexia. Immunol. Investig. 24: 479-487.

Jacob C. O., Holoshitz J., Van Der Meide P., Strober S. and H. O.McDevitt (1989) Heterogeneous effects of IFN-γ in adjuvant arthritis. J.Immunol. 142: 1500-1505.

Jiang H., Milo R., Swoveland P., Johnson KP, Panitch H. and S.Dhib-Jalbut. (1995) Interferon beta-1b reduces interferon gamma-inducedantigen-presenting capacity of human glial and B cells. J. Neuroimmunol.61: 17-25

Kaneko F., Suzuki M., Takiguchi Y., Itoh N. and T. Minagawa (1990)Immunohistopathologic studies in the development of psoriatic lesioninfluenced by gamma-interferon and the producing cells. J. Dermatol.Sci. 1: 425-434.

Kettleborough C. A., Saldanha J., Heath V. J., Morrison C. J. and M. M.Bendig (1991) Humanization of a mouse monoclonal antibody byCDR-grafting: the importance of framework residues on loop conformation.Protein Engineering 4: 773-783.

Kipriyanov S., Little M., Kropshofer H., Breitling F., Gotter S. and S.Dubel (1996) Affinity enhancement of a recombinant antibody: formationof complexes with multiple valency by a single-chain Fv fragment-corestreptavidin fusion. Prot. Eng. 9:203.

Knappik A. and A. Plückthun (1995) Engineered turns of a recombinantantibody improve its in vivo folding. Protein Engineering 8: 81-89.

Kortt A., Lah M., Oddie G., Gruen C., Burns J., Pearce L., Atwell J.,McCoy A., Howlet G., Metzger D., Webster R. and P. Hudson (1997)Single-chain Fv fragments of anti-neuramidase antibody NC10 containingfive- and ten-residue linkers form dimers and with zero-residue linker atrimer. Prot. Eng. 10:423.

Köhler G. and C. Milstein (1975) Continuous cultures of fused cellssecreting antibody of predefined specificity. Nature 256:495.

Kostelny S., Cole M. and Y. Yun Tso (1992) Formation of a bispecificantibody by the use of leucine zippers. J. Immunol. 148:1547.

Kranz D., Gruber M. and E. Wilson (1995) Properties of bispecific singlechain antibodies expressed in E. coli. J. Hematother. 4:403.

Kreutzer B., Stubiger N., Thiel H. J. and M. Zierhut (1996)Oculomucocutaneous changes as paraneoplastic syndromes. Ger. J.Ophtalmol. 5:176-181.

Kwok A. Y. C., Zu X., Yang C., Alfa M. J. and F. T. Jay (1993) Humaninterferon- has three domains associated with its antiviral function: aneutralizing epitope typing scheme for human interferon-γ. Immunology78:131-137.

Landolfo S., Cofano F., Giovarelli M., Pratt M., Cavallo G., and G.Forni (1985) Inhibition of interferon-gamma may suppress allograftreactivity by T lymphocytes in vitro and in vivo. Science 229:176-179.

Langstein H. N., Doherty G. M., Fraker D. L., Buresh C. M. and J. A.Norton (1 991) The roles of γ-interferon and tumor necrosis factor in anexperimental rat model of cancer cachexia. Cancer Research 51:2302-2306.

Lewis J. A. (1995) A sensitive biological assay for interferons. J.Immunol. Meth. 185:9-17.

Lorsbach R. B., Murphy W. J., Lowenstein C. J., Snyder S. H. and S. W.Russel (1993) Expression of the nitric oxide synthase gene in mousemacrophages activated for tumor cell killing. J. Biol. Chem. 268:1908-1913.

Mándi Y., Farkas G., Ocsovszky I. and Z. Nagy (1995) Inhibition of tumornecrosis factor production and ICAM-1 expression by pentoxifylline:beneficial effects in sepsis syndrome. Res. Exp. Med. (Berl) 195:297-307.

Manning L. R., Jenkins W. T., Hess J. R., Vandegriff K., Winslow R. M.and J. M. Manning (1996) Subunit dissociations in natural andrecombinant hemoglobins. Protein Science 5: 775-781.

Massacesi et al. (1995) Active and passively induced experimentalautoimmune encephalomyelitis in common marmosets: a new model formultiple sclerosis. Ann. Neurol. 37: 519-530.

Mateo C., Moreno E., Amour K., Lombardero J., Harris W. and R. Perez(1997) Humanization of a mouse monoclonal antibody that blocks theepidermal growth factor receptor: recovery of antagonistic activity.Immunotechnology 3:71-81.

Matthys P., Dijkmans S., Proost P. et al. (1991) Severe cachexia in miceinoculated with interferon-γ producing tumor cells. Int. J. Cancer49:77-82.

McCarron R. M., Wang L., Racke M. K., Mc Farlin D. E. and M. Spatz(1993) Cytokine-regulated adhesion between encephalitogenic Tlymphocytes and cerebrovascular endothelial cells. J. Neuroimmunol. 43:23-30.

McCutchan J. H. and J. S. Pagano (1968) Enchancement of the infectivityof simian virus 40 deoxyribonucleic acid with diethylaminoethyl-dextran.J-Natl-Cancer-Inst. 41: 351-7.

Megahed M. (1996) Histology of subepidermal bullous dermatoses. Verh.Dtsch. Ges. Pathol. 80:223-228.

Meissner K., Weyer U., Kowalzick L. and J. Altenhoff (1991) Successfultreatment of primary progressive follicular mucinosis with interferons.J. Am. Acad. Dermatol. 24:848-850.

Miethke T., Duschek K., Wahl C, Heeg K. and H. Wagner (1993)Pathogenesis of the toxic shock syndrome: T cell mediated lethal shockcaused by the superantigen TSST-1. Eur. J. Immunol. 23: 1494-1500.

Montero-Julian F. A., B. Klein, E. Gautherot and H. Brailly (1995)Pharmacokinetic study of anti-interleukin-6 (IL-6) clearance by coctailsof anti-IL-6 antibodies. Blood 85: 917-924.

Morel P., Revillard J-P., Nicolas J-F., Vijdenes J., Rizova H. and J.Thivolet (1992) J. Autoimmunity 4:465-477.

Mould R. M., Hoffman O. M. and T. Brittain (1994) Production of humanembryonic haemoglobin (Gower II) in a yeast expression system. Biochem.J. 298: 619-622.

Nepom G. T. (1993) MHC and Autoimmune Diseases. Immunol. Ser. 59:143-164.

Neu H. C. and L. A. Heppel (1965) The release of enzymes fromEscherichia coli by osmotic shock and during the formation ofspheroplasts. J. Biol. Chem. 240:3685-3692.

Nickoloff B. J. (1988) Role of interferon-gamma in cutaneous traffickingof lymphocytes with emphasis on molecular and cellular adhesion events.Arch. Dermatol. 124: 1835-1843.

Novelli F., Giovarelli M., Reber-Liske R., Virgallita G., Garotta G. andG. Forni (1991) Blockade of physiologically secreted IFN-γ inhibitshuman T lymphocyte and natural killer cell activation. J. Immunol.147:1445-1452.

Olerup O. and J. Hillert (1991) HLA class II-associated geneticsusceptibility in multiple sclerosis: a critical evaluation. Tissueantigens 38:1-15.

Olson J. S., Eich R. F., Smith L. P., Warren J. J. and Knowles B. C.(1997) Protein engineering strategies for designing more stablehemoglobin-based blood substitutes. Artif. Cells Blood Substit. Immobil.Biotechnol., 25: 227-241.

Ozmen L., Pericin M., Hakimi J., Chizzonite R. A., Wysocka M.,Trinchieri G, Gately M. and G. Garotta (1994) Interleukin 12, Interferonγ, and Tumor Necrosis Factor α Are the Key Cytokines of the GeneralizedShwartzman Reaction. J. Exp. Med. 180: 907-915.

Ozmen L., Roman D., Fountoulakis M., Schmid G., Ryffel B. And G. Garotta(1995) Experimental therapy of systemic lupus erythematosus: thetreatment of NZB/W mice with mouse soluble interferon-γ receptorinhibits the onset of glomerulonephritis. Eur. J. Immunol. 25: 6-12.

Pace J. L., Russell S. W., Torres B. A., Johnson H. M. and P. W. Gray(1983) Recombinant mouse γ interferon induces the priming step inmacrophage activation for tumor cell killing. J.Immunol. 130: 2011-2013.

Pack P. and A. Plückthun (1992) Miniantibodies : use of amphipathichelices to produce functional, flexibly linked dimeric Fv fragments withhigh avidity in Escherichia coli. Biochemistry 31: 1579-1584.

Pack P., Kujau M., Schroekh V., Knüpfer U., Wenderoth R., Riesenberg D.and A. Plückthun (1993) Improved bivalent miniantibodies, with identicalavidity as whole antibodies, produced by high cell density fermentationof Escherichia coli. Bio/Technology 11: 1271-1277.

Pack P., Müller K., Zahn R. and A. Plückthun (1995) Tetravalentminiantibodies with high avidity assembling in Escherichia coli. J. Mol.Biol. 246: 28-34.

Page M. and Thorpe R. (1996) Purification of IgG using protein A orprotein G. In: The protein protocols handbook. Walker J. M. (Ed.), HumanPress, Totowa, N.J., pp. 733.

Pagnier J., Baudin V. and C. Poyart (1992) Expression of recombinanthuman hemoglobin. Rev. Fr. Transfus. Hemobiol. 35: 407-415.

Panitch H. S. (1994) Influence of infection on exacerbations of multiplesclerosis. Ann. Neurol. 36 (suppl) S25-28

Panitch H. S., Haley A. S., Hirsch R. L. and K. P. Johnson (1986) Atrial of interferon gamma in multiple sclerosis: clinical results.Neurology 36 (suppl. 1): 285

Pantaleeva G. A. (1990) Paraneoplastic bullous dermatoses. Vestn.Dermatol. Venerol. 2:50-52.

Park S. S., Ryu C. J., Gripon P., Guguen-Guillouzo C. and H. J. Hong(1996) Generation and characterization of a humanized antibody withspecificity for preS2 surface antigen of hepatitis B virus. Hybridoma15:435-441.

Perry M. and Kirby H. (1990) Monoclonal antibodies and their fragments.In: Protein purification applications, a practical approach. Harris E.L. V., Angal S. (Eds.), Oxford University Press, Oxford, UK. pp.147-156.

Pettit D. K. and W. R. Gombotz (1998) The development of site-specificdrug-delivery systems for protein and peptide biopharmaceuticals.Tibtech. 16: 343.

Pin S., Royer C. A., Gratton E., Alpert B. and G. Weber (1990) Subunitinteractions in hemoglobin probed by fluorescence and high-pressuretechniques. Biochemistry 29: 9194-9202.

Plückthun A. and P. Pack (1997) New protein engineering approaches tomultivalent and bispecific antibody fragments. Immunotechnology 3:83-105.

Poljak R. J. (1994) Production and structure of diabodies. Structure2:1121-1123.

Rep M. H., Hintzen R. Q., Polman C. H. and R. A. Van Lier (1996)Recombinant interferon-beta blocks proliferation but enhancesinterleukin-10 secretion by activated human T-cells. J. Neuroimmunol.67: 111-118.

Reinhart K., Wiegand-LIohnert C., Grimminger F., Kaul M., Withington S.,Treacher D., Eckart J., Willatts S., Bouza C., Krausch D., StockenhuberF., Eiselstein J., Daum L. and J. Kempeni (1996) Assessment of thesafety and efficacy of the monoclonal anti-tumor necrosis factorantibody-fragment, MAK 195F, in patients with sepsis and septic shock: amulticenter, randomized, placebo-controlled, dose-ranging study. Crit.Care Med. 24: 733-742.

Reuss-Borst M. A., Pawalec G., Saal J. G., Horny H. P., Muller C. A. andH. D. Waller (1993) Sweet's syndrome associated with myelodysplasia:possible role of cytokines in the pathogenesis of the disease. Br. J.Haematol. 84:356-358.

Roguska M. A., Pedersen J. T., Keddy C. A., Henry A. H., Searle S. J.,Lambert J. M., Goldmacher V. S., Blättler W. A., Rees A. R. and B. C.Guild (1994) Humanization of murine monoclonal antibodies throughvariable domain resurfacing. Proc. Natl. Acad. Sci. USA 91: 969-973.

Rubio M. A., Sotillos M., Jochems G., Alvarez V. and A. L. Corbi (1995)Monocyte activation: rapid induction of α1/β1 (VLA-1) integrinexpression by lipopolysaccharide and interferon-. Eur. J. Immunol. 25:2701-2705.

Saha B., Harlan D. M., Lee K. P., June C. H. and R. Abe (1996)Protection Against Lethal Toxic Shock by Targeted Disruption of the CD28Gene. The Journal of Experimental Medicine 183: 2675-2680.

Sandvig S., Laskay T., Andersson J., De Ley M. and U. Andersson (1987)Gamma-interferon is produced by CD3⁺ and CD3³¹ lymphocytes. Immun. Rev.97:51-65.

Schon M. P., Detmar M. and C. M. Parker (1997) Murine psoriasis-likedisorders induced by naive CD4⁺ cells. Nature Medicine 3:183.

Smoller B. R. and J. Bortz (1993) Immunophenotypic analysis suggeststhat granuloma faciale is a gamma-interferon-mediated process. J. Cutan.Pathol. 20:442-446.

Snapper C. M. and W. E. Paul (1987) Interferon-γ and B cell stimulatoryfactor-1 reciprocally regulate Ig isotype production. Science 236:944-947.

Somner N., Loschmann P.-A., Northoff G. H., Weller M., Steinbrecher A.,Steinbach J. P., Lichtenfels R., Meyermann R., Riethmuller A., FontanaA., Dichgans J. and R. Martin (1995) The antidepressant rolipramsuppresses cytokine production and prevents autoimmuneencephalomyelitis. Nature Med. 1: 244-248.

Srere P. A. and Mathews C. K. (1990) Purification of multienzymecomplexes. Methods in Enzymology 182:539-552.

Steinman R. M., Noguiera N., Witmer M. D., Tydings J. G. and I. S.Mellman (1980) Lymphokine enhances the expression and synthesis of Iaantigens on cultural mouse peritoneal macrophages. J. Exp. Med. 152:1248-1261.

Steffen B. J., Butcher E. C. and B. Engelhardt (1994) Evidence forinvolvement of ICAM-1 and VCAM-1 in lymphocyte interaction withendothelium in experimental autoimmune encephalomyelitis in the centralnervous system in SJL/J mouse. Am. J. Pathol. 145: 189-201.

Stemmer W. P., Crameri A., Ha K. D., Brennan T. M. and Heyneker H. L.(1995) Single-step assembly of a gene and entire plasmid from largenumbers of oligodeoxyribonucleotides. Gene 164: 49-53.

Sutherland-Smith A. J., Baker H. M., Hofmann O. M., Brittain T. and E.N. Baker (1998) Crystal structure of a human embryonic haemoglobin: thecarbonmonoxy form of gower II (alpha2epsilon2) haemoglobin at 2.9 Aresolution. J.Mol. Biol. 280: 475-484.

Tang H., Mignon-Godefroy K., Meroni P. L., Garotta G., Charreire J. andF. Nicoletti (1993) The effects of a monoclonal antibody to interferon-γon experimental autoimmune thyroiditis (EAT): prevention of disease anddecrease of EAT-specific T cells. Eur. J. Immunol. 23: 275-278.

Teraki Y., Imanishi K. and T. Shiohara (1996) Ofuji's disease andcytokines: remission of eosinophilic pustular folliculutis associatedwith increased serum concentrations of interferon gamma. Dermatology192:16-18.

Terrell T. G. and J. D. Green (1993) Comparative pathology ofrecombinant murine interferon- in mice and recombinant humaninterferon-γ in cynomolgus monkeys. Int. Rev. Exp. Pathology 34B:73-101.

Terskikh A. V., Le Doussal J-M., Crameri R., Fisch I., Mach J-P. and A.V. Kajava (1997) “Peptabody”: a new type of high avidity bindingprotein. Proc. Natl. Acad. Sci. USA 94: 1663-1668.

Tracey K. J. (1991) Tumor necrosis factor (cachectin) in the biology ofseptic shock syndrome. Circ. Shock 35: 123-128.

Tsukada N., Matsuda M., Miyagi K. and N. Yanagisawa (1993) Cytotoxicityof T cells for cerebral endothelium in multiple sclerosis. J. Neurol.Sci. 117: 140-147.

Turano A., Balsari A., Viani E., Landolfo S., Zanoni L., Gargiulo F. andA. Caruso (1992) Natural human antibodies to γ interferon interfere withthe immunomodulating activity of the lymphokine. Proc. Natl. Acad. Sci.USA 89:4447-4451.

Valdimarsson H., Baker B. S., Jonsdottir I., Powles A. and L. Fry.(1995)Psoriasis: a T-cell-mediated autoimmune disease induced by streptococcalsuperantigens? Immunol. Today 16:145.

Van den Oord J. J., De Ley M. and C. De Wolf-Peeters (1995) Distributionof interferon-gamma receptors in normal and psoriatic skin. Path. Res.Pract. 191:530-534.

Villinger F., Hunt D., Mayne A., Vuchetich M., Findley H. and A. A.Ansari (1993) Qualitative and quantitative studies of cytokinessynthesized and secreted by non-human primate peripheral bloodmononuclear cells. Cytokine 5:469-479.

Vowels B. R., Lessin S. R., Cassin M., Jaworsky C., Benoit B., Wolfe J.T. and A. H. Rook (1994) Th2 cytokine mRNA expression in skin incutaneous T-cell lymphoma. J. Invest. Dermatol. 103:669-673.

Waisman A., P. J. Ruiz, D. L. Hirschberg, A. Gelman, J. R. Oksenberg, S.Brocke, F. Mor, I. R. Cohen and L. Steinman (1996) Suppressivevaccination with DNA encoding a variable region gene of the T-cellreceptor prevents autoimmune encephalomyelitis and activates Th2immunity. Nature Medicine 2: 899-905.

Wakabayashi G., Gelfand J. A., Burke J. F., Thompson R. C. and C. A.Dinarello (1991) A specific receptor antagonist for interleukin-1prevents Escherichia coli-induced septic shock during lethal bacteremia.Nature 330: 662-664.

Waldburger K. E., Hastings R. C., Schaub R. G., Goldman S. J. and J. P.Leonard (1996) Adoptive transfer of experimental allergicencephalomyelitis after in vitro treatment with recombinant murineinterleukin-12. Preferential expansion of interferon-gamma-producingcells and increased expression of macrophage-associated inducible nitricoxide synthase as immunomodulatory mechanisms. Am. J. Pathol. 148:375-82.

Wherry J., Wenzel R., Wunderik R. et al. (1993) Monoclonal antibody tohuman tumor necrosis factor (TNFα MAb): Multicenter efficacy and safetystudy in patients with sepsis syndrome. Presented at the 33thInterscience Conference Antimicrobial Agents and Chemotherapy, NewOrleans; abstract 696.

Willenborg D. O., Fordham S. A., Cowden W. B. and I. A. Ramshaw (1995)Cytokines and murine autoimmune encephalomyelitis: inhibition orenhancement of disease with antibodies to select cytokines, or bydelivery of exogenous cytokines using a recombinant vaccinia virussystem. Scand. J. Immunol. 41: 31-41.

Williams K. C., Ulvestad E. and W. F. Hickey (1994) Immunology ofmultiple sclerosis. Clin. Neurosci. 2: 229-245.

Willsteed E., Bhogal B. S., Das A. K., Wojnarowska F., Black M. M. andP. H. McKee (1991) Lichen planus pemphigoides: a clinicopathologicalstidy of nine cases. Histopathology 19:147-154.

Wood G. S., Michie S. A., Durden F., Hoppe R. T and R. A. Warnke (1994)Expression of class II major histocompatibility antigens bykeratinocytes in cutaneous T cell lymphoma. Int. J. Dermatol.33:346-350.

XuY., Oomen R. and M. H. Klein (1994) Residue at position 331 in theIgG1 and IgG4 CH2 domains contributes to their differential ability tobind and activate complement. J. Biol. Chem. 269:3469-74.

Youl B. D., Turano G., Miller D. H., Towell A. D., MacManus D. G., MooreS. G., Jones S. J., Barrett G., Kendall B. E., Moseley I. F., Tofts P.S., Halliday A. M. and W. I. McDonald (1991) The pathophysiology ofacute optic neuritis. Brain 114: 2437-2450.

Yu C.-L., Haskard D. O., Cavender D., Johnson A. R. and M. Ziff (1985)Human gamma interferon increases binding of T lymphocytes to endothelialcells. Clin. Exp. Immunol. 62: 554-560.

Zeni F., Pain P., Vindimian M; Gay J. P., Gery P., Bertrand M., Page Y.,Vermesch R. and J. C. Bertrand (1996) Effects of pentoxifylline oncirculating cytokine concentrations and hemodynamics in patients withseptic shock: results from a double-blind, randomized,placebo-controlled study. Crit. Care Med. 24: 207-214.

Zhu Z., Zapata G., Shalaby R., Snedecor B., Chen H. and P. Carter (1996)High level secretion of a humanized bispecific diabody from Escherichiacoli. Biotechnology 14:192-196.

104 1 804 DNA UNKNOWN GENOMIC 1 atgaaatacc tattgcctac ggcagccgctggattgttat tactcgctgc ccaaccagcg 60 atggcccagg tgcagctggt gcagagcggtagcgaactga aaaaaccggg tgcgagcgtt 120 aagatcagct gcaaagcgag cggttataccttcaccgatt acggtatgaa ctgggttaaa 180 caggcgccgg gtcaaggtct gaaatggatgggttggatca acacctacac cggtgaaagc 240 acctacgttg acgatttcaa aggtcgtttcgttttcagcc tggataccag cgttagcgcg 300 gcctacctgc agatcagctc tctgaaagcggaagacaccg cgacctactt ctgcgcgcgt 360 cgcggtttct acgcgatgga ttactggggccaagggacca cggtcaccgt ctcctcaggt 420 ggaggcggtt caggcggagg tggctctggcggtggcggat cggacatcgt actgacccag 480 agcccggcga ccatgagcgc gagcccgggtgaacgtgtta ccctgacctg cagcgcgagc 540 tctagcatca gctatatgtt ctggtatcatcagcgtccgg gtcagagccc gcgtctgttg 600 atctatgata ccagcaacct ggcgagcggtgttccggcgc gtttcagcgg tagcggtagc 660 ggtaccagct atagcctgac catcagccgtatggaaccgg aagatttcgc gacctatttc 720 tgccatcaga gctctagcta tccgttcaccttcggtcagg gtaccaaact cgagatcaaa 780 cggcaccatc accatcacca ctaa 804 2267 PRT Artificial Sequence Synthetic 2 Met Lys Tyr Leu Leu Pro Thr AlaAla Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala GlnVal Gln Leu Val Gln Ser Gly Ser Glu 20 25 30 Leu Lys Lys Pro Gly Ala SerVal Lys Ile Ser Cys Lys Ala Ser Gly 35 40 45 Tyr Thr Phe Thr Asp Tyr GlyMet Asn Trp Val Lys Gln Ala Pro Gly 50 55 60 Gln Gly Leu Lys Trp Met GlyTrp Ile Asn Thr Tyr Thr Gly Glu Ser 65 70 75 80 Thr Tyr Val Asp Asp PheLys Gly Arg Phe Val Phe Ser Leu Asp Thr 85 90 95 Ser Val Ser Ala Ala TyrLeu Gln Ile Ser Ser Leu Lys Ala Glu Asp 100 105 110 Thr Ala Thr Tyr PheCys Ala Arg Arg Gly Phe Tyr Ala Met Asp Tyr 115 120 125 Trp Gly Gln GlyThr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly GlyGly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln 145 150 155 160 SerPro Ala Thr Met Ser Ala Ser Pro Gly Glu Arg Val Thr Leu Thr 165 170 175Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met Phe Trp Tyr His Gln Arg 180 185190 Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Asp Thr Ser Asn Leu Ala 195200 205 Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr210 215 220 Ser Leu Thr Ile Ser Arg Met Glu Pro Glu Asp Phe Ala Thr TyrPhe 225 230 235 240 Cys His Gln Ser Ser Ser Tyr Pro Phe Thr Phe Gly GlnGly Thr Lys 245 250 255 Leu Glu Ile Lys Arg His His His His His His 260265 3 40 DNA UNKNOWN GENOMIC 3 cgcgcagccg ctggattgtt attactcgctgcccaaccag 40 4 40 DNA UNKNOWN GENOMIC 4 cagctgcacc tgggccatcgctggttgggc agcgagtaat 40 5 40 DNA UNKNOWN GENOMIC 5 cgatggcccaggtgcagctg gtgcagagcg gtagcgaact 40 6 40 DNA UNKNOWN GENOMIC 6cgctcgcacc cggttttttc agttcgctac cgctctgcac 40 7 40 DNA UNKNOWN GENOMIC7 gaaaaaaccg ggtgcgagcg ttaagatcag ctgcaaagcg 40 8 40 DNA UNKNOWNGENOMIC 8 tcggtgaagg tataaccgct cgctttgcag ctgatcttaa 40 9 40 DNAUNKNOWN GENOMIC 9 agcggttata ccttcaccga ttacggtatg aactgggtta 40 10 40DNA UNKNOWN GENOMIC 10 accttgaccc ggcgcctgtt taacccagtt cataccgtaa 40 1140 DNA UNKNOWN GENOMIC 11 aacaggcgcc gggtcaaggt ctgaaatgga tgggttggat 4012 40 DNA UNKNOWN GENOMIC 12 tttcaccggt gtaggtgttg atccaaccca tccatttcag40 13 40 DNA UNKNOWN GENOMIC 13 caacacctac accggtgaaa gcacctacgttgacgatttc 40 14 40 DNA UNKNOWN GENOMIC 14 ctgaaaacga aacgacctttgaaatcgtca acgtaggtgc 40 15 40 DNA UNKNOWN GENOMIC 15 aaaggtcgtttcgttttcag cctggatacc agcgttagcg 40 16 40 DNA UNKNOWN GENOMIC 16gctgatctgc aggtaggccg cgctaacgct ggtatccagg 40 17 40 DNA UNKNOWN GENOMIC17 cggcctacct gcagatcagc tctctgaaag cggaagacac 40 18 40 DNA UNKNOWNGENOMIC 18 gcgcgcagaa gtaggtcgcg gtgtcttccg ctttcagaga 40 19 40 DNAUNKNOWN GENOMIC 19 cgcgacctac ttctgcgcgc gtcgcggttt ctacgcgatg 40 20 41DNA UNKNOWN GENOMIC 20 gcgcccttgg ccccagtaat ccatcgcgta gaaaccgcga c 4121 25 DNA UNKNOWN GENOMIC 21 cgcgcagccg ctggattgtt attac 25 22 21 DNAUNKNOWN GENOMIC 22 gcgcccttgg ccccagtaat c 21 23 48 DNA UNKNOWN GENOMIC23 cgcggtatac tgacccagag cccggcgacc atgagcgcga gcccgggt 48 24 40 DNAUNKNOWN GENOMIC 24 caggtcaggg taacacgttc acccgggctc gcgctcatgg 40 25 40DNA UNKNOWN GENOMIC 25 gaacgtgtta ccctgacctg cagcgcgagc tctagcatca 40 2640 DNA UNKNOWN GENOMIC 26 atgataccag aacatatagc tgatgctaga gctcgcgctg 4027 40 DNA UNKNOWN GENOMIC 27 gctatatgtt ctggtatcat cagcgtccgg gtcagagccc40 28 40 DNA UNKNOWN GENOMIC 28 tatcatagat caacagacgc gggctctgacccggacgctg 40 29 40 DNA UNKNOWN GENOMIC 29 gcgtctgttg atctatgataccagcaacct ggcgagcggt 40 30 40 DNA UNKNOWN GENOMIC 30 ccgctgaaacgcgccggaac accgctcgcc aggttgctgg 40 31 40 DNA UNKNOWN GENOMIC 31gttccggcgc gtttcagcgg tagcggtagc ggtaccagct 40 32 40 DNA UNKNOWN GENOMIC32 acggctgatg gtcaggctat agctggtacc gctaccgcta 40 33 40 DNA UNKNOWNGENOMIC 33 atagcctgac catcagccgt atggaaccgg aagatttcgc 40 34 40 DNAUNKNOWN GENOMIC 34 tctgatggca gaaataggtc gcgaaatctt ccggttccat 40 35 40DNA UNKNOWN GENOMIC 35 gacctatttc tgccatcaga gctctagcta tccgttcacc 40 3648 DNA UNKNOWN GENOMIC 36 cgcgctcgag tttggtaccc tgaccgaagg tgaacggatagctagagc 48 37 21 DNA UNKNOWN GENOMIC 37 cgcggtatac tgacccagag c 21 3822 DNA UNKNOWN GENOMIC 38 cgcgctcgag tttggtaccc tg 22 39 26 DNA UNKNOWNGENOMIC 39 tcgagatcaa acggtaatag ccatgg 26 40 26 DNA UNKNOWN GENOMIC 40aattccatgg ctattaccgt ttgatc 26 41 32 DNA UNKNOWN GENOMIC 41 tcgaagcttagtactgtggc tgcaccatct gt 32 42 32 DNA UNKNOWN GENOMIC 42 gtcgaattctgcgcactctc ccctgttgaa gc 32 43 48 DNA UNKNOWN GENOMIC 43 ctagaattctgcgcatccac caagggccca tcggtcttcc ccctggca 48 44 36 DNA UNKNOWN GENOMIC44 gtaaagcttg agctcttacc cggagacagg gagagg 36 45 40 DNA UNKNOWN GENOMIC45 gtcccccggg tacctctaga atggattttc aagtgcagat 40 46 40 DNA UNKNOWNGENOMIC 46 tttcagcttc ctgctaatca gtgcctcagt catactctcg 40 47 40 DNAUNKNOWN GENOMIC 47 ctctgggtca gctcgatgtc cgagagtatg actgaggcac 40 48 40DNA UNKNOWN GENOMIC 48 tgattagcag gaagctgaaa atctgcactt gaaaatccat 40 4923 DNA UNKNOWN GENOMIC 49 gtcccccggg tacctctaga atg 23 50 21 DNA UNKNOWNGENOMIC 50 ctctgggtca gctcgatgtc c 21 51 27 DNA UNKNOWN GENOMIC 51gacatcgagc tgacccagag cccggcg 27 52 22 DNA UNKNOWN GENOMIC 52 cgcgctcgagtttggtaccc tg 22 53 38 DNA UNKNOWN GENOMIC 53 gcgcctcgag atcaaacggactgtggctgc accatctg 38 54 32 DNA UNKNOWN GENOMIC 54 gccggaattcctagcactct cccctgttga ag 32 55 40 DNA UNKNOWN GENOMIC 55 ctctgcaccagctgcacctg cgagagtatg actgaggcac 40 56 21 DNA UNKNOWN GENOMIC 56ctctgcacca gctgcacctg c 21 57 26 DNA UNKNOWN GENOMIC 57 caggtgcagctggtgcagag cggtag 26 58 45 DNA UNKNOWN GENOMIC 58 cgccggctcg agacggtgaccgtggtccct tggccccagt aatcc 45 59 21 DNA UNKNOWN GENOMIC 59 cgccggctcgagacggtgac c 21 60 26 DNA UNKNOWN GENOMIC 60 gccgctcgag cgcatccaccaagggc 26 61 39 DNA UNKNOWN GENOMIC 61 gccggaattc gctaaagctt acccggagacagggagagg 39 62 26 DNA UNKNOWN GENOMIC 62 gccctcccag cctccatcga gaaaac26 63 26 DNA UNKNOWN GENOMIC 63 gttttctcga tggaggctgg gagggc 26 64 15DNA UNKNOWN GENOMIC 64 taatacgact cacta 15 65 18 DNA UNKNOWN GENOMIC 65atttaggtga cactatag 18 66 1404 DNA UNKNOWN GENOMIC 66 atggattttcaagtgcagat tttcagcttc ctgctaatca gtgcctcagt catactctcg 60 caggtgcagctggtgcagag cggtagcgaa ctgaaaaaac cgggtgcgag cgttaagatc 120 agctgcaaagcgagcggtta taccttcacc gattacggta tgaactgggt taaacaggcg 180 ccgggtcaaggtctgaaatg gatgggttgg atcaacacct acaccggtga aagcacctac 240 gttgacgatttcaaaggtcg tttcgttttc agcctggata ccagcgttag cgcggcctac 300 ctgcagatcagctctctgaa agcggaagac accgcgacct acttctgcgc gcgtcgcggt 360 ttctacgcgatggattactg gggccaaggg accacggtca ccgtctcgag cgcatccacc 420 aagggcccatcggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 480 gccctgggctgcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540 ggcgccctgaccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 600 tccctcagcagcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 660 aacgtgaatcacaagcccag caacaccaag gtggacaaga gagttgagcc caaatcttgt 720 gacaaaactcacacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 780 ttcctcttccccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 840 tgcgtggtggtggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 900 ggcgtggaggtgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 960 cgtgtggtcagcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 1020 tgcaaggtctccaacaaagc cctcccagcc tccatcgaga aaaccatctc caaagccaaa 1080 gggcagccccgagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 1140 aaccaggtcagcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1200 tgggagagcaatgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1260 gacggctccttcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg 1320 aacgtcttctcatgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1380 ctctccctgtctccgggtaa gctt 1404 67 468 PRT Artificial Sequence SYNTHETIC 67 Met AspPhe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 ValIle Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys 20 25 30 LysPro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr 35 40 45 PheThr Asp Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly 50 55 60 LeuLys Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Ser Thr Tyr 65 70 75 80Val Asp Asp Phe Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val 85 90 95Ser Ala Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala 100 105110 Thr Tyr Phe Cys Ala Arg Arg Gly Phe Tyr Ala Met Asp Tyr Trp Gly 115120 125 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser130 135 140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly ThrAla 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu ProVal Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val HisThr Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu SerSer Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr TyrIle Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val Asp LysArg Val Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys ProPro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro Ser Val Phe LeuPhe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr ProGlu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro GluVal Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn AlaLys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320 ArgVal Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile 340 345350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355360 365 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala ValGlu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys ThrThr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr SerLys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val PheSer Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr GlnLys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys Leu 465 68 699 DNAUNKNOWN GENOMIC 68 atggattttc aagtgcagat tttcagcttc ctgctaatcagtgcctcagt catactctcg 60 gacatcgagc tgacccagag cccggcgacc atgagcgcgagcccgggtga acgtgttacc 120 ctgacctgca gcgcgagctc tagcatcagc tatatgttctggtatcatca gcgtccgggt 180 cagagcccgc gtctgttgat ctatgatacc agcaacctggcgagcggtgt tccggcgcgt 240 ttcagcggta gcggtagcgg taccagctat agcctgaccatcagccgtat ggaaccggaa 300 gatttcgcga cctatttctg ccatcagagc tctagctatccgttcacctt cggtcagggt 360 accaaactcg agatcaaacg gactgtggct gcaccatctgtcttcatctt cccgccatct 420 gatgagcagt tgaaatctgg aactgcctct gttgtgtgcctgctgaataa cttctatccc 480 agagaggcca aagtacagtg gaaggtggat aacgccctccaatcgggtaa ctcccaggag 540 agtgtcacag agcaggacag caaggacagc acctacagcctcagcagcac cctgacgctg 600 agcaaagcag actacgagaa acacaaagtc tacgcctgcgaagtcaccca tcagggcctg 660 agctcgcccg tcacaaagag cttcaacagg ggagagtgc 69969 233 PRT Artificial Sequence SYNTHETIC 69 Met Asp Phe Gln Val Gln IlePhe Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Leu Ser Asp IleGlu Leu Thr Gln Ser Pro Ala Thr Met Ser 20 25 30 Ala Ser Pro Gly Glu ArgVal Thr Leu Thr Cys Ser Ala Ser Ser Ser 35 40 45 Ile Ser Tyr Met Phe TrpTyr His Gln Arg Pro Gly Gln Ser Pro Arg 50 55 60 Leu Leu Ile Tyr Asp ThrSer Asn Leu Ala Ser Gly Val Pro Ala Arg 65 70 75 80 Phe Ser Gly Ser GlySer Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg 85 90 95 Met Glu Pro Glu AspPhe Ala Thr Tyr Phe Cys His Gln Ser Ser Ser 100 105 110 Tyr Pro Phe ThrPhe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr 115 120 125 Val Ala AlaPro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 130 135 140 Lys SerGly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 145 150 155 160Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 165 170175 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180185 190 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His195 200 205 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser ProVal 210 215 220 Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 70 41 DNAUNKNOWN GENOMIC 70 cgcgctcgag atcaaacgga ccccgctggg tgataccact c 41 7140 DNA UNKNOWN GENOMIC 71 cagttcacct ccggaggtat gagtggtatc acccagcggg 4072 40 DNA UNKNOWN GENOMIC 72 atacctccgg aggtgaactg gaagagctgt tgaaacatct40 73 40 DNA UNKNOWN GENOMIC 73 gacctttcag cagttctttc agatgtttcaacagctcttc 40 74 40 DNA UNKNOWN GENOMIC 74 gaaagaactg ctgaaaggtccgcggaaagg tgaactggag 40 75 40 DNA UNKNOWN GENOMIC 75 ttcaggtgcttcagcaattc ctccagttca cctttccgcg 40 76 40 DNA UNKNOWN GENOMIC 76gaattgctga agcacctgaa agagctgttg aaaggtaccc 40 77 40 DNA UNKNOWN GENOMIC77 atgggtagta tcacctaggg gggtaccttt caacagctct 40 78 40 DNA UNKNOWNGENOMIC 78 ccctaggtga tactacccat accagcggtc aggtgcaact 40 79 42 DNAUNKNOWN GENOMIC 79 cgcggaattc gcgttcgcga ctagttgcac ctgaccgctg gt 42 8021 DNA UNKNOWN GENOMIC 80 cgcggtatac tgacccagag c 21 81 22 DNA UNKNOWNGENOMIC 81 cgcgctcgag tttggtaccc tg 22 82 29 DNA UNKNOWN GENOMIC 82cgcgactagt gcagagcggt agcgaactg 29 83 26 DNA UNKNOWN GENOMIC 83gccagtgaat tctattagtg gtgatg 26 84 1626 DNA UNKNOWN GENOMIC 84caggtgcagc tggtgcagag cggtagcgaa ctgaaaaaac cgggtgcgag cgttaagatc 60agctgcaaag cgagcggtta taccttcacc gattacggta tgaactgggt taaacaggcg 120ccgggtcaag gtctgaaatg gatgggttgg atcaacacct acaccggtga aagcacctac 180gttgacgatt tcaaaggtcg tttcgttttc agcctggata ccagcgttag cgcggcctac 240ctgcagatca gctctctgaa agcggaagac accgcgacct acttctgcgc gcgtcgcggt 300ttctacgcga tggattactg gggccaaggg accacggtca ccgtctcctc aggtggaggc 360ggttcaggcg gaggtggctc tggcggtggc ggatcggaca tcgtactgac ccagagcccg 420gcgaccatga gcgcgagccc gggtgaacgt gttaccctga cctgcagcgc gagctctagc 480atcagctata tgttctggta tcatcagcgt ccgggtcaga gcccgcgtct gttgatctat 540gataccagca acctggcgag cggtgttccg gcgcgtttca gcggtagcgg tagcggtacc 600agctatagcc tgaccatcag ccgtatggaa ccggaagatt tcgcgaccta tttctgccat 660cagagctcta gctatccgtt caccttcggt cagggtacca aactcgagat caaacggacc 720ccgctgggtg ataccactca tacctccgga ggtgaactgg aagagctgtt gaaacatctg 780aaagaactgc tgaaaggtcc gcggaaaggt gaactggagg aattgctgaa gcacctgaaa 840gagctgttga aaggtacccc cctgggtgat actacccata ccagcggtca ggtgcaacta 900gtgcagagcg gtagcgaact gaaaaaaccg ggtgcgagcg ttaagatcag ctgcaaagcg 960agcggttata ccttcaccga ttacggtatg aactgggtta aacaggcgcc gggtcaaggt 1020ctgaaatgga tgggttggat caacacctac accggtgaaa gcacctacgt tgacgatttc 1080aaaggtcgtt tcgttttcag cctggatacc agcgttagcg cggcctacct gcagatcagc 1140tctctgaaag cggaagacac cgcgacctac ttctgcgcgc gtcgcggttt ctacgcgatg 1200gattactggg gccaagggac cacggtcacc gtctcctcag gtggaggcgg ttcaggcgga 1260ggtggctctg gcggtggcgg atcggacatc gtactgaccc agagcccggc gaccatgagc 1320gcgagcccgg gtgaacgtgt taccctgacc tgcagcgcga gctctagcat cagctatatg 1380ttctggtatc atcagcgtcc gggtcagagc ccgcgtctgt tgatctatga taccagcaac 1440ctggcgagcg gtgttccggc gcgtttcagc ggtagcggta gcggtaccag ctatagcctg 1500accatcagcc gtatggaacc ggaagatttc gcgacctatt tctgccatca gagctctagc 1560tatccgttca ccttcggtca gggtaccaaa ctcgagatca aacggcacca tcaccatcac 1620cactaa 1626 85 541 PRT Artificial Sequence SYNTHETIC 85 Gln Val Gln LeuVal Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val LysIle Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Gly Met AsnTrp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45 Gly Trp IleAsn Thr Tyr Thr Gly Glu Ser Thr Tyr Val Asp Asp Phe 50 55 60 Lys Gly ArgPhe Val Phe Ser Leu Asp Thr Ser Val Ser Ala Ala Tyr 65 70 75 80 Leu GlnIle Ser Ser Leu Lys Ala Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala ArgArg Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 ValThr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Met Ser 130 135140 Ala Ser Pro Gly Glu Arg Val Thr Leu Thr Cys Ser Ala Ser Ser Ser 145150 155 160 Ile Ser Tyr Met Phe Trp Tyr His Gln Arg Pro Gly Gln Ser ProArg 165 170 175 Leu Leu Ile Tyr Asp Thr Ser Asn Leu Ala Ser Gly Val ProAla Arg 180 185 190 Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu ThrIle Ser Arg 195 200 205 Met Glu Pro Glu Asp Phe Ala Thr Tyr Phe Cys HisGln Ser Ser Ser 210 215 220 Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys LeuGlu Ile Lys Arg Thr 225 230 235 240 Pro Leu Gly Asp Thr Thr His Thr SerGly Gly Glu Leu Glu Glu Leu 245 250 255 Leu Lys His Leu Lys Glu Leu LeuLys Gly Pro Arg Lys Gly Glu Leu 260 265 270 Glu Glu Leu Leu Lys His LeuLys Glu Leu Leu Lys Gly Thr Pro Leu 275 280 285 Gly Asp Thr Thr His ThrSer Gly Gln Val Gln Leu Val Gln Ser Gly 290 295 300 Ser Glu Leu Lys LysPro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala 305 310 315 320 Ser Gly TyrThr Phe Thr Asp Tyr Gly Met Asn Trp Val Lys Gln Ala 325 330 335 Pro GlyGln Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly 340 345 350 GluSer Thr Tyr Val Asp Asp Phe Lys Gly Arg Phe Val Phe Ser Leu 355 360 365Asp Thr Ser Val Ser Ala Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala 370 375380 Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Arg Gly Phe Tyr Ala Met 385390 395 400 Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly GlyGly 405 410 415 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp IleVal Leu 420 425 430 Thr Gln Ser Pro Ala Thr Met Ser Ala Ser Pro Gly GluArg Val Thr 435 440 445 Leu Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr MetPhe Trp Tyr His 450 455 460 Gln Arg Pro Gly Gln Ser Pro Arg Leu Leu IleTyr Asp Thr Ser Asn 465 470 475 480 Leu Ala Ser Gly Val Pro Ala Arg PheSer Gly Ser Gly Ser Gly Thr 485 490 495 Ser Tyr Ser Leu Thr Ile Ser ArgMet Glu Pro Glu Asp Phe Ala Thr 500 505 510 Tyr Phe Cys His Gln Ser SerSer Tyr Pro Phe Thr Phe Gly Gln Gly 515 520 525 Thr Lys Leu Glu Ile LysArg His His His His His His 530 535 540 86 3423 DNA UNKNOWN GENOMIC 86ttccggggat ctctcaccta ccaaacaatg cccccctgca aaaaataaat tcatataaaa 60aacatacaga taaccatctg cggtgataaa ttatctctgg cggtgttgac ataaatacca 120ctggcggtga tactgagcac atcagcagga cgcactgacc accatgaagg tgacgctctt 180aaaaattaag ccctgaagaa gggcaggggt accaggaggt ttaaatcatg gtaagatcaa 240gtagtcaaaa ttcgagtgac aagcctgtag cccacgtcgt agcaaaccac caagtggagg 300agcagtaacc atggttactg gagaaggggg accaactcag cgctgaggtc aatctgccca 360agtctagagt cgacctgcag cccaagcttg gctgttttgg cggatgagag aagattttca 420gcctgataca gattaaatca gaacgcagaa gcggtctgat aaaacagaat ttgcctggcg 480gcagtagcgc ggtggtccca cctgacccca tgccgaactc agaagtgaaa cgccgtagcg 540ccgatggtag tgtggggtct ccccatgcga gagtagggaa ctgccaggca tcaaataaaa 600cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct 660ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga 720gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca gaaggccatc 780ctgacggatg gcctttttgc gtttctacaa actcttttgt ttatttttct aaatacattc 840aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat aaaaggatct 900aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc 960actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 1020gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 1080atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa 1140atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 1200ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 1260gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 1320cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 1380tacagcgtga gcattgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 1440cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 1500ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 1560gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc 1620tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg 1680ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc 1740gcagcgagtc agtgagcgag gaagcggaag agcgctgact tccgcgtttc cagactttac 1800gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag 1860cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc 1920cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggccaggac 1980ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga tggcggacgc gatggatatg 2040ttctgccaag ggttggtttg cgcattcaca gttctccgca agaattgatt ggctccaatt 2100cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc gaggtggccc 2160ggctccatgc accgcgacgc aacgcgggga ggcagacaag gtatagggcg gcgcctacaa 2220tccatgccaa cccgttccat gtgctcgccg aggcggcata aatcgccgtg acgatcagcg 2280gtccagtgat cgaagttagg ctggtaagag ccgcgagcga tccttgaagc tgtccctgat 2340ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc gggcatcccg atgccgccgg 2400aagcgagaag aatcataatg gggaaggcca tccagcctcg cgtcgcgaac gccagcaaga 2460cgtagcccag cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt 2520tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa 2580gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga 2640gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga 2700cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca 2760tcggtcggcg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga 2820ggccgttgag caccgccgcc gcaaggaatg gtgcatgtaa ggagatggcg cccaacagtc 2880ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt 2940ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg 3000tggcgccggt gatgccggcc acgatgcgtc cggcgtagag aatccacagg acgggtgtgg 3060tcgccatgat cgcgtagtcg atagtggctc caagtagcga agcgagcagg actgggcggc 3120ggccaaagcg gtcggacagt gctccgagaa cgggtgcgca tagaaattgc atcaacgcat 3180atagcgctag cagcacgcca tagtgactgg cgatgctgtc ggaatggacg atatcccgca 3240agaggcccgg cagtaccggc ataaccaagc ctatgcctac agcatccagg gtgacggtgc 3300cgaggatgac gatgagcgca ttgttagatt tcatacacgg tgcctgactg cgttagcaat 3360ttaactgtga taaactaccg cattaaagct aatcgatgat aagctgtcaa acatgagaat 3420taa 3423 87 41 DNA UNKNOWN GENOMIC 87 cccaagcttg gcggaggctc acaggtgcagctggtgcaga g 41 88 30 DNA UNKNOWN GENOMIC 88 cggaattcta ccgtttgatctcgagtttgg 30 89 2133 DNA UNKNOWN GENOMIC 89 atggattttc aagtgcagattttcagcttc ctgctaatca gtgcctcagt catactctcg 60 caggtgcagc tggtgcagagcggtagcgaa ctgaaaaaac cgggtgcgag cgttaagatc 120 agctgcaaag cgagcggttataccttcacc gattacggta tgaactgggt taaacaggcg 180 ccgggtcaag gtctgaaatggatgggttgg atcaacacct acaccggtga aagcacctac 240 gttgacgatt tcaaaggtcgtttcgttttc agcctggata ccagcgttag cgcggcctac 300 ctgcagatca gctctctgaaagcggaagac accgcgacct acttctgcgc gcgtcgcggt 360 ttctacgcga tggattactggggccaaggg accacggtca ccgtctcgag cgcatccacc 420 aagggcccat cggtcttccccctggcaccc tcctccaaga gcacctctgg gggcacagcg 480 gccctgggct gcctggtcaaggactacttc cccgaaccgg tgacggtgtc gtggaactca 540 ggcgccctga ccagcggcgtgcacaccttc ccggctgtcc tacagtcctc aggactctac 600 tccctcagca gcgtggtgaccgtgccctcc agcagcttgg gcacccagac ctacatctgc 660 aacgtgaatc acaagcccagcaacaccaag gtggacaaga gagttgagcc caaatcttgt 720 gacaaaactc acacatgcccaccgtgccca gcacctgaac tcctgggggg accgtcagtc 780 ttcctcttcc ccccaaaacccaaggacacc ctcatgatct cccggacccc tgaggtcaca 840 tgcgtggtgg tggacgtgagccacgaagac cctgaggtca agttcaactg gtacgtggac 900 ggcgtggagg tgcataatgccaagacaaag ccgcgggagg agcagtacaa cagcacgtac 960 cgtgtggtca gcgtcctcaccgtcctgcac caggactggc tgaatggcaa ggagtacaag 1020 tgcaaggtct ccaacaaagccctcccagcc tccatcgaga aaaccatctc caaagccaaa 1080 gggcagcccc gagaaccacaggtgtacacc ctgcccccat cccgggagga gatgaccaag 1140 aaccaggtca gcctgacctgcctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1200 tgggagagca atgggcagccggagaacaac tacaagacca cgcctcccgt gctggactcc 1260 gacggctcct tcttcctctatagcaagctc accgtggaca agagcaggtg gcagcagggg 1320 aacgtcttct catgctccgtgatgcatgag gctctgcaca accactacac gcagaagagc 1380 ctctccctgt ctccgggtaagcttggcgga ggctcacagg tgcagctggt gcagagcggt 1440 agcgaactga aaaaaccgggtgcgagcgtt aagatcagct gcaaagcgag cggttatacc 1500 ttcaccgatt acggtatgaactgggttaaa caggcgccgg gtcaaggtct gaaatggatg 1560 ggttggatca acacctacaccggtgaaagc acctacgttg acgatttcaa aggtcgtttc 1620 gttttcagcc tggataccagcgttagcgcg gcctacctgc agatcagctc tctgaaagcg 1680 gaagacaccg cgacctacttctgcgcgcgt cgcggtttct acgcgatgga ttactggggc 1740 caagggacca cggtcaccgtctcctcaggt ggaggcggtt caggcggagg tggctctggc 1800 ggtggcggat cggacatcgtactgacccag agcccggcga ccatgagcgc gagcccgggt 1860 gaacgtgtta ccctgacctgcagcgcgagc tctagcatca gctatatgtt ctggtatcat 1920 cagcgtccgg gtcagagcccgcgtctgttg atctatgata ccagcaacct ggcgagcggt 1980 gttccggcgc gtttcagcggtagcggtagc ggtaccagct atagcctgac catcagccgt 2040 atggaaccgg aagatttcgcgacctatttc tgccatcaga gctctagcta tccgttcacc 2100 ttcggtcagg gtaccaaactcgagatcaaa cgg 2133 90 711 PRT Artificial Sequence SYNTHETIC 90 Met AspPhe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 ValIle Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys 20 25 30 LysPro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr 35 40 45 PheThr Asp Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly 50 55 60 LeuLys Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Ser Thr Tyr 65 70 75 80Val Asp Asp Phe Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val 85 90 95Ser Ala Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala 100 105110 Thr Tyr Phe Cys Ala Arg Arg Gly Phe Tyr Ala Met Asp Tyr Trp Gly 115120 125 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser130 135 140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly ThrAla 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu ProVal Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val HisThr Phe Pro Ala 180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu SerSer Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr TyrIle Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val Asp LysArg Val Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys ProPro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro Ser Val Phe LeuPhe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr ProGlu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro GluVal Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn AlaLys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320 ArgVal Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 325 330 335Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile 340 345350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355360 365 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala ValGlu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys ThrThr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr SerLys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val PheSer Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr GlnLys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys Leu Gly Gly Gly Ser GlnVal Gln Leu Val Gln Ser Gly 465 470 475 480 Ser Glu Leu Lys Lys Pro GlyAla Ser Val Lys Ile Ser Cys Lys Ala 485 490 495 Ser Gly Tyr Thr Phe ThrAsp Tyr Gly Met Asn Trp Val Lys Gln Ala 500 505 510 Pro Gly Gln Gly LeuLys Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly 515 520 525 Glu Ser Thr TyrVal Asp Asp Phe Lys Gly Arg Phe Val Phe Ser Leu 530 535 540 Asp Thr SerVal Ser Ala Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala 545 550 555 560 GluAsp Thr Ala Thr Tyr Phe Cys Ala Arg Arg Gly Phe Tyr Ala Met 565 570 575Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly 580 585590 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu 595600 605 Thr Gln Ser Pro Ala Thr Met Ser Ala Ser Pro Gly Glu Arg Val Thr610 615 620 Leu Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met Phe Trp TyrHis 625 630 635 640 Gln Arg Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr AspThr Ser Asn 645 650 655 Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly SerGly Ser Gly Thr 660 665 670 Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu ProGlu Asp Phe Ala Thr 675 680 685 Tyr Phe Cys His Gln Ser Ser Ser Tyr ProPhe Thr Phe Gly Gln Gly 690 695 700 Thr Lys Leu Glu Ile Lys Arg 705 71091 240 PRT Artificial Sequence SYNTHETIC 91 Gln Val Gln Leu Val Gln SerGly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser CysLys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Gly Met Asn Trp Val LysGln Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr TyrThr Gly Glu Ser Thr Tyr Val Asp Asp Phe 50 55 60 Lys Gly Arg Phe Val PheSer Leu Asp Thr Ser Val Ser Ala Ala Tyr 65 70 75 80 Leu Gln Ile Ser SerLeu Lys Ala Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Arg Gly PheTyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val SerSer Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 115 120 125 Ile Val LeuThr Gln Ser Pro Ala Thr Met Ser Ala Ser Pro Gly Glu 130 135 140 Arg ValThr Leu Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met Phe 145 150 155 160Trp Tyr His Gln Arg Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Asp 165 170175 Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly 180185 190 Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Pro Glu Asp195 200 205 Phe Ala Thr Tyr Phe Cys His Gln Ser Ser Ser Tyr Pro Phe ThrPhe 210 215 220 Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg His His His HisHis His 225 230 235 240 92 702 DNA UNKNOWN GENOMIC 92 caggtgcagctggtgcagag cggtagcgaa ctgaaaaaac cgggtgcgag cgttaagatc 60 agctgcaaagcgagcggtta taccttcacc gattacggta tgaactgggt taaacaggcg 120 ccgggtcaaggtctgaaatg gatgggttgg atcaacacct acaccggtga aagcacctac 180 gttgacgatttcaaaggtcg tttcgttttc agcctggata ccagcgttag cgcggcctac 240 ctgcagatcagctctctgaa agcggaagac accgcgacct acttctgcgc gcgtcgcggt 300 ttctacgcgatggattactg gggccaaggg accacggtca ccgtctcctc aggcggaggt 360 ggctctggcggtggcggatc ggacatcgta ctgacccaga gcccggcgac catgagcgcg 420 agcccgggtgaacgtgttac cctgacctgc agcgcgagct ctagcatcag ctatatgttc 480 tggtatcatcagcgtccggg tcagagcccg cgtctgttga tctatgatac cagcaacctg 540 gcgagcggtgttccggcgcg tttcagcggt agcggtagcg gtaccagcta tagcctgacc 600 atcagccgtatggaaccgga agatttcgcg acctatttct gccatcagag ctctagctat 660 ccgttcaccttcggtcaggg taccaaactc gagatcaaac gg 702 93 235 PRT Artificial SequenceSYNTHETIC 93 Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro GlyAla 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe ThrAsp Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu LysTrp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Ser Thr Tyr Val AspAsp Phe 50 55 60 Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser AlaAla Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala ThrTyr Phe Cys 85 90 95 Ala Arg Arg Gly Phe Tyr Ala Met Asp Tyr Trp Gly GlnGly Thr Thr 100 105 110 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp IleVal Leu Thr Gln 115 120 125 Ser Pro Ala Thr Met Ser Ala Ser Pro Gly GluArg Val Thr Leu Thr 130 135 140 Cys Ser Ala Ser Ser Ser Ile Ser Tyr MetPhe Trp Tyr His Gln Arg 145 150 155 160 Pro Gly Gln Ser Pro Arg Leu LeuIle Tyr Asp Thr Ser Asn Leu Ala 165 170 175 Ser Gly Val Pro Ala Arg PheSer Gly Ser Gly Ser Gly Thr Ser Tyr 180 185 190 Ser Leu Thr Ile Ser ArgMet Glu Pro Glu Asp Phe Ala Thr Tyr Phe 195 200 205 Cys His Gln Ser SerSer Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys 210 215 220 Leu Glu Ile LysArg His His His His His His 225 230 235 94 687 DNA UNKNOWN GENOMIC 94caggtgcagc tggtgcagag cggtagcgaa ctgaaaaaac cgggtgcgag cgttaagatc 60agctgcaaag cgagcggtta taccttcacc gattacggta tgaactgggt taaacaggcg 120ccgggtcaag gtctgaaatg gatgggttgg atcaacacct acaccggtga aagcacctac 180gttgacgatt tcaaaggtcg tttcgttttc agcctggata ccagcgttag cgcggcctac 240ctgcagatca gctctctgaa agcggaagac accgcgacct acttctgcgc gcgtcgcggt 300ttctacgcga tggattactg gggccaaggg accacggtca ccgtctcctc aggcggtggc 360ggatcggaca tcgtactgac ccagagcccg gcgaccatga gcgcgagccc gggtgaacgt 420gttaccctga cctgcagcgc gagctctagc atcagctata tgttctggta tcatcagcgt 480ccgggtcaga gcccgcgtct gttgatctat gataccagca acctggcgag cggtgttccg 540gcgcgtttca gcggtagcgg tagcggtacc agctatagcc tgaccatcag ccgtatggaa 600ccggaagatt tcgcgaccta tttctgccat cagagctcta gctatccgtt caccttcggt 660cagggtacca aactcgagat caaacgg 687 95 34 DNA UNKNOWN GENOMIC 95ggccgctctt cgaaatacct attgcctacg gcag 34 96 70 DNA UNKNOWN GENOMIC 96ctgggtcagt acgatgtcag agccacctcc gcctgaaccg cctccacctg aggagacggt 60gaccgtggtc 70 97 67 DNA UNKNOWN GENOMIC 97 gtcaccgtct cctcaggtggaggcggttca ggcggaggtg gctctgacat cgtactgacc 60 cagagcc 67 98 26 DNAUNKNOWN GENOMIC 98 gccagtgaat tctattagtg gtgatg 26 99 55 DNA UNKNOWNGENOMIC 99 ctgggtcagt acgatgtctg aaccgcctcc acctgaggag acggtgaccg tggtc55 100 52 DNA UNKNOWN GENOMIC 100 gtcaccgtct cctcaggtgg aggcggttcagacatcgtac tgacccagag cc 52 101 672 DNA UNKNOWN GENOMIC 101 caggtgcagctggtgcagag cggtagcgaa ctgaaaaaac cgggtgcgag cgttaagatc 60 agctgcaaagcgagcggtta taccttcacc gattacggta tgaactgggt taaacaggcg 120 ccgggtcaaggtctgaaatg gatgggttgg atcaacacct acaccggtga aagcacctac 180 gttgacgatttcaaaggtcg tttcgttttc agcctggata ccagcgttag cgcggcctac 240 ctgcagatcagctctctgaa agcggaagac accgcgacct acttctgcgc gcgtcgcggt 300 ttctacgcgatggattactg gggccaaggg accacggtca ccgtctcctc agacatcgta 360 ctgacccagagcccggcgac catgagcgcg agcccgggtg aacgtgttac cctgacctgc 420 agcgcgagctctagcatcag ctatatgttc tggtatcatc agcgtccggg tcagagcccg 480 cgtctgttgatctatgatac cagcaacctg gcgagcggtg ttccggcgcg tttcagcggt 540 agcggtagcggtaccagcta tagcctgacc atcagccgta tggaaccgga agatttcgcg 600 acctatttctgccatcagag ctctagctat ccgttcacct tcggtcaggg taccaaactc 660 gagatcaaac gg672 102 230 PRT Artificial Sequence SYNTHETIC 102 Gln Val Gln Leu ValGln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys IleSer Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Gly Met Asn TrpVal Lys Gln Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile AsnThr Tyr Thr Gly Glu Ser Thr Tyr Val Asp Asp Phe 50 55 60 Lys Gly Arg PheVal Phe Ser Leu Asp Thr Ser Val Ser Ala Ala Tyr 65 70 75 80 Leu Gln IleSer Ser Leu Lys Ala Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg ArgGly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Val ThrVal Ser Ser Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Met 115 120 125 SerAla Ser Pro Gly Glu Arg Val Thr Leu Thr Cys Ser Ala Ser Ser 130 135 140Ser Ile Ser Tyr Met Phe Trp Tyr His Gln Arg Pro Gly Gln Ser Pro 145 150155 160 Arg Leu Leu Ile Tyr Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Ala165 170 175 Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr IleSer 180 185 190 Arg Met Glu Pro Glu Asp Phe Ala Thr Tyr Phe Cys His GlnSer Ser 195 200 205 Ser Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu GluIle Lys Arg 210 215 220 His His His His His His 225 230 103 40 DNAUNKNOWN GENOMIC 103 ctgggtcagt acgatgtctg aggagacggt gaccgtggtc 40 10437 DNA UNKNOWN GENOMIC 104 gtcaccgtct cctcagacat cgtactgacc cagagcc 37

What is claimed is:
 1. An isolated molecule which binds and neutralizesinterferon-gamma selected from the group consisting of: a scFvcomprising a humanized variable domain, wherein said variable domaincomprises amino acids 1-117 and 133-239 of SEQ ID) NO: 85; a chimericantibody comprising: a) a humanized heavy chain variable domain, saidheavy chain variable domain having an amino acid sequence as shown inpositions 1-117 of SEQ ID NO: 85, and b) the humanized light chainvariable domain, said light chain variable domain having an amino acidsequence as shown in positions 133-239 of SEQ ID NO: 85; a diabodycomprising: a) a humanized heavy chain variable domain, said heavy chainvariable domain having an amino acid sequence as shown in positions1-117 of SEQ ID NO: 85, and b) a humanized light chain variable domain,said light chain variable domain having an amino acid sequence as shownin positions 133-239 of SEQ ID NO: 85; and, a multivalent antibody,wherein said multivalent antibody is selected from the group consistingof a triabody, a tetravalent antibody, a peptabody, and a hexabody, andwherein said multivalent antibody comprises: a) a humanized heavy chainvariable domain, said variable domain comprising amino acids 1-117 ofSEQ ID NO: 85; and b) a humanized light chain variable domain, saidvariable domain comprising amino acids 133-239 of SEQ ID NO:
 85. 2. Anisolated molecule according to claim 1, wherein said triabody furthercomprises: a) three variable domains of three differentanti-interferon-gamma antibodies, or b) at least one variable domain ofan anti-interferon-gamma antibody in combination with i) at least onevariable domain of a different anti-interferon-gamma antibody, or ii) atleast one variable domain of an antibody which binds to another moleculeexcluding interferon-gamma; wherein at least one of the variable domainscomprises amino acids 1-117 and 133-239 of SEQ ID NO:85.
 3. The isolatedmolecule according to claim 1, which is a triabody further comprisingthree identical variable domains of an anti-interferon-gamma antibody.4. The isolated molecule according to claim 1, which is a triabodyfurther comprising three identical humanized scFvs, wherein each scFvhas a zero residue linker joining the humanized heavy chain variabledomain to the humanized light chain variable domain.
 5. The isolatedmolecule according to claim 1, which is a tetravalent antibody furthercomprising: a) four variable domains of four differentanti-interferon-gamma antibodies, or b) at least one variable domain ofan anti-interferon-gamma antibody in combination with i) at least onevariable domain of another anti-interferon-gamma antibody, or ii) anantibody which binds to another molecule excluding interferon gamma;wherein at least one of the variable domains comprises amino acids 1-117and 133-239 of SEQ ID NO:85.
 6. The isolated molecule according to claim1, which is a tetravalent antibody further comprising four identicalvariable domains of an anti-interferon-gamma antibody.
 7. The isolatedmolecule according to claim 1, which is a tetravalent antibody furthercomprising four identical humanized scFvs as a homodimer of twoidentical molecules, each containing two humanized scFvs and adimerization domain.
 8. The isolated molecule according to claim 7,wherein each said scFv comprises amino acids 1-239 of SEQ ID NO:
 85. 9.The isolated molecule according to claim 1, which is a tetravalentantibody further comprising: a) a full-sized humanized antibody whereinsaid antibody comprises two heavy chains and two light chains, and b)two humanized scFvs wherein each scFv is attached by itscarboxy-terminus to a carboxy-terminus of one of said antibody's heavychains, and wherein each said scFv comprises amino acids 1-239 of SEQ IDNO:
 85. 10. The isolated molecule according to claim 1, which is eithera peptabody comprising five identical variable domains of ananti-interferon-gamma antibody, or a hexabody comprising six identicalvariable domains of an anti-interferon-gamma antibody.
 11. The isolatedmolecule according to claim 1, which is either a peptabody comprisingfive identical humanized scFvs, or a hexabody comprising six identicalhumanized scFvs.
 12. The isolated molecule according to claim 11,wherein each said scFv comprises amino acids 1-239 of SEQ ID NO:
 85. 13.The isolated molecule according to claim 1, which is either a) apeptabody comprising a combination of 1 to 4 variable domains from ananti-interferon-gamma antibody and, respectively, 4 to 1 variabledomain(s) of an antibody which binds to another molecule other thaninterferon gamma, wherein at least one of the variable domains comprisesamino acids 1-117 and 133-239 of SEQ ID NO:85; or b) a hexabodycomprising a combination of 1 to 5 variable domains from ananti-interferon-gamma antibody and, respectively, 5 to 1 variabledomain(s) of an antibody which binds to another molecule other thaninterferon gamma, wherein at least one of the variable domains comprisesamino acids 1-117 and 133-239 of SEQ ID NO:85.
 14. The moleculeaccording to claim 1, which is either: a) a peptabody comprising fivevariable domains from five different anti-interferon-gamma antibodies,wherein at least one of the variable domains comprises amino acids 1-117and 133-239 of SEQ ID NO:85; or b) a hexabody comprising six variabledomains from six different anti-interferon-gamma antibodies, wherein atleast one of the variable domains comprises amino acids 1-117 and133-239 of SEQ ID NO:85.